Macroscopic Changes Observed after Burning in Incised Wounds on Bones Caused by Sharp and Penetrating Instruments: A Preliminary Experimental Study on Bovine Bones

Background: Stab and incised wounds are frequently encountered in forensic practice. Burned skeletal remains often complicate the evaluation of such injuries, making it essential to understand how fire alters cut marks on bones. This study aimed to investigate macroscopic changes in bones subjected to sharp-force trauma after exposure to high temperatures. Methods: Three bovine humeri were incised with a 32 cm non-serrated kitchen knife. The length, width, direction, and interrelationships of each incision were measured with a forensic scale, photographed, and recorded. The bones were then burned in open-air conditions using oak wood, without accelerants, at 460–570 °C for 60 minutes. Following combustion, the incisions were remeasured, and pre- and post-burning characteristics were compared statistically. Results: Despite advanced thermal destruction, most incisions remained macroscopically visible. Heat exposure caused significant increases in incision length and width (p < 0.05). Linear fissures radiating from the cuts were observed, and the cut margins appeared sharper and drier after burning. In one specimen, thermal fractures disrupted the evaluation of a distal incision. Conclusion: Sharp-force trauma marks on bones are not completely obscured by fire and may even become more pronounced. However, heat-related expansion of cut dimensions can lead to overestimation of blade size. These findings emphasize the need for caution in interpreting burned skeletal remains and highlight the importance of further research with larger sample sizes to improve forensic accuracy

نقش مشارکت ورزشی بر نشاط اجتماعی و بهره¬وری سازمانی کارکنان شرکت آذین الکترونیک پیشرو

مقدمه: هدف از پژوهش حاضر بررسی نقش مشارکت ورزشی بر نشاط اجتماعی و بهره­وری سازمانی کارکنان شرکت آذین الکترونیک پیشرو بود. روش کار: روش پژوهش حاضر توصیفی از نوع همبستگی، بر اساس زمان حال­نگر، از نظر هدف کاربردی و به لحاظ جمع­آوری اطلاعات میدانی بود. جامعة آماري پژوهش شامل همه کارکنانی هستند که در سال 1404 در شرکت آذین الکترونیک پیشرو تهران فعالیت داشتند که تعداد کل آن­ها بنا بر آمار اعلام شده از سوی شرکت الکترونیک پیشرو 500 نفر بودند. نمونه آماری طبق جدول مورگان تعداد 217 نفر از بين جامعه آماري بودند که به صورت در دسترس انتخاب شدند. از پرسشنامه­های مشارکت ورزشی گیل، گروس و هادلستون (1983)، نشاط اجتماعی تمیزی فر و عزیزی مهر (1396) و بهره­وری سازمانی شریفی (1391) برای اندازه­گیری متغیرهای تحقیق استفاده شد. یافته­ها: مشارکت ورزشی بر نشاط اجتماعی و بهره­وری سازمانی کارکنان شرکت آذین الکترونیک پیشرو نقش داشت. نتیجه­گیری: بنابراین با کمک مشارکت ورزشی می­توان منجر به افزایش نشاط اجتماعی و بهره­وری سازمانی کارکنان شرکت آذین الکترونیک پیشرو شد. لذا پیشنهاد می­شود که مدیران به وضعیت نشاط اجتماعی و بهره­وری سازمانی در کارکنان توجه بیشتری نمایند تا از طریق مشارکت ورزشی به نشاط اجتماعی و بهره­وری سازمانی کمک نمایند

Multicystic Dysplastic Kidney Disease: A Review: Multicystic Dysplastic Kidney

The cystic kidney is one of the most common abnormalities detectable by ultrasound beforeand after birth and is also a cause of hydronephrosis; therefore, ultrasound is necessary foraccurate diagnosis. Multicystic dysplastic kidney (MCDK) is a congenital disease in whichthe kidney is filled with multiple cysts and does not function properly. This article describesan article review of MCDK and new attention. This study aimed to increase awareness ofthe importance of diseases that cause kidney cysts, especially dysplastic multicystic kidneydisease, to promote understanding of diagnostic methods before and after birth, and toinvestigate its cause

تأثیر هشت هفته تمرینات سیکلیک یوگا بر سندرم متقاطع فوقانی، لوردوز افزایش یافته و سلامت روانی زنان سالمند

Background and Aim: Postural abnormalities and Upper Crossed Syndrome are common problems among elderly women that can lead to physical and psychological disorders. The aim of the present study was to investigate the effect of eight weeks of cyclic yoga exercises on Upper Crossed Syndrome and mental health in elderly women. Methods: 30 elderly women over 60 years old with postural abnormalities including thoracic kyphosis, forward head, and lumbar lordosis were purposefully selected and randomly divided into two experimental and control groups (15 people in each group). This research was conducted with ethical approval code IR.SHAHROODUT.REC.1403.008 from the Ethics Committee of Shahrood University of Technology, and ethical considerations such as informed consent, voluntary participation, and confidentiality of information were observed. The experimental group participated in selected cyclic yoga exercises for eight weeks, three sessions per week, each session lasting 45 to 90 minutes. A flexible ruler was used to measure thoracic kyphosis and lumbar lordosis, a goniometer for forward head angle, and the General Health Questionnaire (GHQ) to assess mental health status. Data were analyzed using paired t-test and analysis of covariance in SPSS software version 22. Results: The results showed that cyclic yoga exercises led to a significant reduction in thoracic kyphosis angle, forward head angle, lumbar lordosis angle, and a significant improvement in mental health in the exercise group compared to the control group. Conclusion: Cyclic yoga exercises can be used as an effective intervention to improve skeletal-muscular abnormalities and promote mental health in elderly women. Please cite this article as: Soltanian T. Rezvani MH, Naderi A, ChoromZadeh H. Effect of Eight Weeks of Cyclic Yoga Practice on Upper Crossed Syndrome, Increased lordosis and Mental Health in Elderly Women. Irtiqa Imini Pishgiri Masdumiyat .2024;12(4):248-259. https://doi.org/10.22037/iipm.v11i3.49200سابقه و هدف: ناهنجاری‌های وضعیتی و سندرم متقاطع فوقانی از مشکلات شایع در میان زنان سالمند هستند که می‌توانند منجر به اختلالات جسمانی و روانی شوند. هدف پژوهش حاضر بررسی تأثیر هشت هفته تمرینات سیکلیک یوگا بر سندرم متقاطع فوقانی و سلامت روانی در زنان سالمند بود. روش کار: ۳۰ زن سالمند بالای ۶۰ سال با ناهنجاری‌های وضعیتی شامل کیفوز پشتی، سر به جلو و لوردوز کمری به‌صورت هدفمند انتخاب و به‌طور تصادفی در دو گروه آزمایش و کنترل (هر گروه ۱۵ نفر) تقسیم شدند. این پژوهش با اخذ تأییدیه اخلاقی با کد IR.SHAHROODUT.REC.1403.008  از کمیته اخلاق دانشگاه صنعتی شاهرود انجام شد و ملاحظات اخلاقی نظیر رضایت آگاهانه، مشارکت داوطلبانه و محرمانگی اطلاعات رعایت گردید. گروه آزمایش به مدت هشت هفته، سه جلسه در هفته و هر جلسه به مدت ۴۵ تا ۹۰ دقیقه در تمرینات منتخب سیکلیک یوگا شرکت کردند. برای سنجش کیفوز پشتی و لوردوز کمری از خط‌کش منعطف، برای زاویه سر به جلو از گونیامتر و برای بررسی وضعیت سلامت روانی از پرسش‌نامه سلامت عمومی GHQ استفاده شد. داده‌ها با استفاده از آزمون تی زوجی و تحلیل کوواریانس در نرم‌افزار SPSS نسخه ۲۲ تحلیل شدند. یافته­ ها: نتایج نشان داد که تمرینات سیکلیک یوگا منجر به کاهش معنادار زاویه کیفوز پشتی، زاویه سر به جلو، زاویه لوردوز کمری و بهبود معنادار سلامت روانی در گروه تمرین نسبت به گروه کنترل شد. نتیجه­ گیری: تمرینات سیکلیک یوگا می‌تواند به‌عنوان یک مداخله مؤثر برای بهبود ناهنجاری‌های اسکلتی‌ـ‌عضلانی و ارتقاء سلامت روانی در زنان سالمند مورد استفاده قرار گیرد. به این مقاله، به صورت زیر استناد کنید: Soltanian T. Rezvani MH, Naderi A, ChoromZadeh H. Effect of Eight Weeks of Cyclic Yoga Practice on Upper Crossed Syndrome, Increased lordosis and Mental Health in Elderly Women. Irtiqa Imini Pishgiri Masdumiyat .2024;12(4):248-259. https://doi.org/10.22037/iipm.v11i3.4920

Fluoride in Focus: How Dentists’ Demographics, Practice Settings, and Perceptions Shape Caries Prevention Strategies

Objective(s): Dental caries remains a prevalent chronic disease globally, with fluoride being a cornerstone in its prevention. Despite its proven efficacy, the implementation of fluoride-based strategies depends heavily on dentists' knowledge, attitudes, and practices. This study assessed the knowledge, attitudes, and practices of Iranian dentists concerning fluoride application, as well as the barriers to its use. Methods: A cross-sectional study was conducted among a convenient sample of 296 dentists. A validated questionnaire was used to collect data on dentists’ age, gender, dental specialty, working sectors and queries, such as the availability of specific guidelines for prescribing fluoride products in the workplace, self-reported practices regarding fluoride application, perceptions of barriers to fluoride use and level of agreement with statements regarding the effectiveness of fluoride in caries prevention.  Descriptive statistics and chi-square tests were employed to summarize and compare responses based on demographic and professional characteristics at p<0.05. Results: 40% of dentists had access to fluoride prescription guidelines. Younger dentists and those in both public and private sectors were more likely to report guideline availability. While 73-97% of dentists agreed with the effectiveness of fluoride in caries prevention, only 50% recognized its benefits for individuals over 12 years old. Fluoride application was more prevalent for children under 12 (30-35%), compared to adults (10%). Barriers to fluoride use included low treatment tariffs (45%), skepticism about its effectiveness (42%), and time constraints (25%). Dentists favored restorative treatments for high-risk cases, with 80% recommending fluoride varnish and gel for children but fewer for adults. Conclusion: This study highlighted gaps in knowledge and practice regarding use of fluoride among Iranian dentists, particularly for adult patients. Barriers such as low reimbursement rates and skepticism about fluoride's effectiveness need to be addressed. Enhanced training, standardized guidelines, and policy interventions are essential to improve fluoride application efforts in Iran

Effectiveness of peer group-based psychosocial empowerment training on high-risk behaviors in male secondary school Students at risk for behavioral issues

Background: Adolescence is a critical developmental stage where high-risk behaviors can jeopardize well-being and future outcomes. This study evaluated the efficacy of peer group-based psychosocial empowerment training in reducing high-risk behaviors among male secondary school students at risk for behavioral issues in Tehran, Iran. Methods: This quasi-experimental study employed a two-group, pre-test, post-test, and three-month follow-up design with a control group. A purposeful sample of 40 at-risk male students, identified through teacher reports and a high-risk behaviors questionnaire, was randomly assigned to a peer-led experimental group (n=20) or a wait-listed control group (n=20). The experimental group received eight 90-minute sessions of psychosocial empowerment training facilitated by trained peers. Data were collected using a validated high-risk behaviors questionnaire (42 items, seven subscales). Repeated measures ANOVA and Bonferroni post-hoc tests were used to analyze group differences over time, with effect sizes (η²) reported. Results: The peer-led intervention significantly reduced high-risk behaviors, including cigarette/hookah use, alcohol/substance consumption, aggressive behaviors, suicidal ideation/attempts, running away, and opposite-sex relationships (F=59.22, P<0.001, η²=0.61, 95% CI [14.63, 22.83] for total score). These reductions were sustained at the three-month follow-up, indicating a robust effect of the intervention on vulnerable adolescents. Conclusion: This study demonstrates the efficacy of peer group-based psychosocial empowerment training as an effective strategy for reducing high-risk behaviors among at-risk male adolescents. The findings support the integration of peer-led interventions into school-based mental health programs to enhance resilience and promote positive youth development

Exploring how core beliefs shape risky self-injury in teens: the bridge of emotional control challenges

Background: Teen years represent a pivotal phase of growth, fraught with distinct risks like tendencies toward self-injurious actions. Our research explored the straightforward connection between core life principles and such behaviors among teens, while probing how challenges in managing feelings act as an intermediary in this dynamic. Methods: Employing a correlative design with structural equation modeling, we targeted Tehran high schoolers aged 14–18 from the 2023–2024 school term. Through multi-phase clustered random selection, 356 youths joined, filling out tools like the Self-Harm Inventory, abbreviated Portrait Values Questionnaire, and compact Difficulties in Emotion Regulation Scale. We crunched numbers via SPSS 27.0 and AMOS. Results: The proposed framework aligned well with real-world findings. As predicted, stronger life principles linked inversely to self-injury (β= 0.11, P=0.044). Emotional control issues tied positively to these behaviors (β=0.37, P=0.001). Notably, feeling management woes bridged part of the gap between principles and self-injury (β=-0.06, P=0.007). Conclusion: This study highlights that personal values, emphasizing meaning, purpose, and prosocial behaviors (e.g., benevolence, universalism), protect against adolescent self-harm by enhancing emotion regulation. Integrating values-based interventions and emotion regulation training into prevention programs is recommended for clinicians and educators to reduce self-harm risks effectively

رویکرد ابن ‏سینا به غربالگری و ارزیابی پیش از فعالیت ورزشی

Background and Aim: Since one of the primary goals of physical activity is to maintain and improve health and enhance the quality of life, achieving this goal requires determining a suitable and safe physical activity for each individual. The American College of Sports Medicine and the American Heart Association have established criteria for screening and assessing individuals' health status and physical fitness levels before prescribing physical activity. Given the valuable status of exercise in maintaining and promoting health in Iranian medicine, this study is the first to investigate Avicenna's approach on assessment and screening before physical activity. Methods: This research was conducted by reviewing Avicenna's book "The Canon of Medicine" and performing an electronic search using relevant keywords. Ethical Considerations: In compiling this study, the principles of trustworthiness, citation integrity, authenticity of sources and avoidance of bias in referring to texts or analyses were observed. Results: Avicenna considers the examination of four factors essential before recommending physical activity: Medical History Review: In chronic diseases, pressure should not be applied to the weak organ. Assessment of Physical Weakness and Strength: Strenuous exercises should not be prescribed for individuals with a weak constitution. Determination of Temperament: Individuals with a warm and dry temperament may be harmed by performing certain exercises. Examination of Signs of Bodily Fullness (Plethora): In individuals whose bodies are filled with waste materials due to an improper lifestyle, exercising before cleansing the body of these materials can be harmful. Conclusion: According to Avicenna's approach, prescribing physical activity should be done after careful assessment. Specific groups such as children, the elderly and patients require special considerations, which aligns with modern research. The two topics of temperament and bodily fullness can provide insightful research ideas for pre-activity screening and assessment, especially since, based on the principles of Iranian medicine, intense physical activity in a state of fullness may lead to sudden cardiac death.زمینه و هدف: با توجه به اینکه یکی از اهداف اصلی فعالیت ورزشی، حفظ و بهبود سلامت و ارتقای کیفیت زندگی است، دستیابی به این هدف مستلزم تعیین فعالیت ورزشی مناسب و ایمن برای هر فرد می‌باشد. کالج پزشکی ورزشی آمریکا و انجمن قلب آمریکا، معیارهایی برای غربالگری و ارزیابی سطح سلامت و آمادگی جسمانی افراد پیش از تجویز فعالیت ورزشی تعیین کرده‌اند. با عنایت به جایگاه ارزشمند ورزش در حفظ و ارتقای سلامت در طب ایرانی، این مطالعه برای نخستین بار به بررسی دیدگاه ابن ‌سینا در مورد ارزیابی و غربالگری پیش از فعالیت ورزشی می‌پردازد. روش: این پژوهش با مرور کتاب «قانون» ابن ‌سینا و جستجوی الکترونیکی با استفاده از کلیدواژه‌های مرتبط انجام شده است. ملاحظات اخلاقی: در تدوین این مطالعه، اصل امانتداری و صداقت استناد به متون مورد استفاده، اصالت منابع و پرهیز از جانبداری در مراجعه به متون یا تحلیل‌ها، رعایت شده است. یافته‌ها: ابن ‏سینا پیش از توصیه به فعالیت ورزشی، بررسی چهار مورد را ضروری می‏داند: 1- بررسی سوابق بیماری: در بیماری‏های مزمن، نباید به عضو ضعیف فشار وارد کرد. 2- ارزیابی میزان ضعف و قوت (بُنیه) بدنی: برای افراد دارای بنیه ضعیف نباید ورزش‏های سنگین تجویز شود. 3- تعیین مزاج: ممکن است افراد با مزاج گرم و خشک از انجام برخی از ورزش‏ها آسیب ببینند. 4- بررسی علایم امتلاء بدنی: در اشخاصی که به دلیل سبک زندگی نادرست، بدنشان انباشته از مواد زاید است، ورزش کردن قبل از پاکسازی بدن می‌تواند مضر باشد. نتیجه‌گیری: بر اساس رویکرد ابن ‌سینا تجویز فعالیت ‌ورزشی باید بعد از ارزیابی دقیق انجام شود. گروه‏های خاص مانند کودکان، سالمندان و بیماران، نیازمند توجهات خاص هستند که همسو با پژوهش‏های نوین است. دو مبحث مزاج و امتلاء بدنی می‏توانند در غربالگری و ارزیابی پیش از فعالیت ورزشی ایده‏های پژوهشی راهگشایی باشند؛ به ویژه با توجه به این که بر اساس مبانی طب ایرانی، فعالیت شدید بدنی در حالت امتلاء ممکن است به مرگ ناگهانی قلبی منجر شود

Purification and Antimicrobial Use of Egg-white Lysozyme against Staphylococcus aureus

Background and Objective: Staphylococcus aureus is a significant foodborne and zoonotic pathogen. This study aimed to enhance the anti-Staphylococcus aureus activity of egg-white lysozyme through heat treatment and synergistic combinations with natural antimicrobials. Material and Methods: The lysozyme was purified from egg white via ammonium sulfate precipitation and cation-exchange chromatography, yielding a homogeneous protein. Anti-Staphylococcus aureus activity of native lysozyme, heat-treated lysozyme and its combination with ferulic acid or Mycobacterium smegmatis acyltransferase was assessed, respectively. All experiments were carried out in triplicate and statistical analysis was carried out using SPSS software. Results and Conclusion: The specific activity of lysozyme to Micrococcus lysodeikticus was 27,407.4 U.mg-1. The lysozyme IC50 against Staphylococcus aureus was 300.8 µg.ml-1, with transmission electron microscopy verifying bacteriolytic action. Heat treatment under optimized conditions (90°C, 15 min, pH 6.2) significantly enhanced the lysozyme antibacterial activity by 35.1%, which was correlated with structural changes evidenced by circular dichroism spectroscopy. Furthermore, synergistic effects were observed when heat-treated lysozyme was combined with ferulic acid or Mycobacterium smegmatis acyltransferase (MsAcT), leading to prolonged inhibition and decreased viable bacterial counts. The findings of this research demonstrated that structural modifications and combinatorial strategies could effectively improve the efficacy and application potential of lysozyme as a natural antimicrobial agent in food safety. Keywords: Egg-white lysozyme, Ferulic acid, Heat-induced fibrillar aggregates, Mycobacterium smegmatis acyltransferase, Staphylococcus aureus Introduction   Staphylococcus aureus is a Gram-positive bacterium widely spread in nature and a common foodborne pathogen. The enterotoxins (SEs) of the bacteria show significant heat resistance, rendering them difficult to completely eliminate through conventional cooking methods and posing a significant risk of food poisoning. Furthermore, S. aureus facilitates cross-infection between humans and animals via the food chain through contamination of animal feed, subsequent infection of animals and transmission to humans, establishing it as an important zoonotic pathogen. The S. aureus has been detected in various animal species and a wide range of food products, with its prevalence continuously increasing on a global scale. Therefore, S. aureus is still a high-priority target in food safety monitoring [1, 2]. To combat S. aureus contamination in food processing, diverse biological control strategies have been investigated. These include inhibition using lactic acid bacteria (LAB) probiotics [3], Lysostaphin [4], bacteriophages [5,6], essential oils (EO) [5] and lysozyme [7,8]. From these, egg-white lysozyme has widely been used due to its high catalytic activity, simple preparation and cost-effectiveness [7,9]. It inhibits S. aureus through two primary mechanisms of (1) lytic mechanism as lysozyme hydrolyzses β-1, 4-glycosidic bonds to peptidoglycan, which causes cell wall damage, induces cell lysis and results in bactericidal activity; and (2) the non-lytic mechanism, where under denaturing conditions, it suppresses growth through inherent protein characteristics such as hydrophobicity and cationic effects [8,10]. While the antimicrobial characteristics of native egg-white lysozyme are well-documented, its efficacy under common food processing conditions, particularly those involving heat, needs further investigation. Moreover, strategies to enhance its activity, especially in a heat-treated state, through combination with other natural antimicrobial agents are still under-investigation. Under various reaction conditions, the interactions between other natural antibacterial agents and egg-white lysozyme can lead to various effects on its antimicrobial activity. For example, in an alkaline solution, theaflavin covalently binds to egg-white lysozyme, resulting in significant decrease of its antibacterial activity [11]. However, in an amyloid fibril hydrogel, the interaction between epigallocatechin gallate (EGCG) and egg-white lysozyme significantly broadens the antibacterial spectrum of egg-white lysozyme [12]. Therefore, an in-depth investigation into the interaction conditions between natural antibacterial factors and egg-white lysozyme is greatly important for enhancing the antibacterial efficiency of egg-white lysozyme. This research aimed to isolate and purify egg-white lysozyme using chromatography and to assess the effect of heat treatment on its anti-S. aureus activity. Furthermore, the study prepared a synergistic combination of heat-treated lysozyme with ferulic acid and acyltransferase to develop an enhanced strategy for suppressing S. aureus growth. The experimental results demonstrated that heat-induced structural modification (fibrillar aggregation) enhanced lysozyme antimicrobial mechanism beyond native peptidoglycan hydrolysis. Moreover, it has first been reported that a synergistic combination of heat-treated lysozyme with ferulic acid and MsAcT against S. aureus significantly improves the efficacy and time of inhibition. Materials and Methods 2.1. Strains, biochemical reagents and chemical reagents Micrococcus lysodeikticus CGMCC 1.4547 and S. aureus CGMCC 1.282 were purchased from China General Microbiological Culture Collection Center, China. Bovine serum albumin (BSA) and standard protein molecular weight marker were purchased from Takara Biomedical Technology, China. Moreover, CM-Sepharose fast flow chromatography column was purchased from GE Healthcare, China. Ferulic acid, caffeic acid, gallic acid and N-acetylglucosamine (NAG) included analytical grade unless otherwise specified and purchased from Shanghai Macklin Biochemical Technology, China. 2.2. Purification of egg-white lysozyme Egg white was initially diluted by 50-fold using 50 mmol.l-1 pH8.0 tris-HCl buffer and the resulting protein solution was ultrasonicated for 10 min. The supernatant was collected by centrifugation at 12,000 rpm for 5 min at 4 °C. Solid ammonium sulfate was added to the supernatant to achieve 40% (w/v) saturation and the mixture was set to precipitate for 2 h at 4 °C. After centrifugation, the precipitate was removed and the collected supernatant was further loaded onto a CM-Sepharose fast flow column (5 × 20 cm) that was pre-equilibrated with 50 mmol.l-1 tris-HCl buffer (pH 8.5). The lysozyme was eluted with a 5-fold column volume of 50 mmol.l-1 tris-HCl buffer (pH 8.5) with increasing concentrations of NaCl (0.1 and 0.5 mol.l-1) at a flow rate of 20 ml.h-1, respectively. The active fractions were pooled. Protein concentration was assessed using Bradford method with BSA as a standard. The homogeneity of the purified egg-white lysozyme was assessed using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 2.3. Activity assay of egg-white lysozyme The cell lysis activity of lysozyme from egg white to M. lysodeikticus was quantitatively assessed using spectrophotometeric assay method, which was described by the National Standard of the People's Republic of China (GB/T 30990-2014, Determination of Lysozyme Acyivity) as well as Naveed et al. [13]. Briefly, after incubation overnight, M. lysodeikticus was transferred into the fresh LB liquid media at 1% (v/v) and incubated at 37 °C for 2 h at 220 rpm. The cells were harvested by centrifugation and resuspended in 50 mmol.l-1 Na2HPO4-Na2HPO4 buffer (pH 6.2) to a final concentration of 5 × 105 CFU.ml-1. The reaction system contained 2.5 ml of cell suspension and 0.5 ml of lysozyme solution, while the control system received 0.5 ml of inactivated lysozyme solution. The reaction was carried out at 50 mmol.l-1 Na2HPO4-Na2HPO4 buffer (pH 6.2) for 5 min at 25 °C and the absorbance of the reaction system was continuously monitored at 450 nm. One unit of lysozyme activity (U) was defined as the quantity of enzyme necessary to decrease OD450 by 0.001 per minute under the standard assay condition. 2.4. Anti-Staphylococcus aureus activity of egg-white lysozyme Briefly, S. aureus in the logarithmic growth phase was used as an indicator microorganism to assess antibacterial activity of the purified lysozyme. To set the growth curve, overnight-cultured S. aureus was subcultured into the fresh LB liquid media at a 1% (v/v) inoculum and incubated at 37 °C for 14 h at 220 rpm. Samples were collected every 30 min to measure the optical density (OD) of the culture broth at 600 nm. The growth curve was plotted with OD600 on the y-axis and incubation time on the x-axis. The antibacterial assessments were carried out using a method described by Carrillo et al. [14] with slight modification. Following 2 h of subculture, S. aureus suspensions were adjusted to 5 × 105 CFU.ml-1 through serial dilution in LB liquid media. Aliquots of S. aureus suspensions were mixed with equal volumes of the purified lysozyme at various concentrations and incubated at 37 °C for 6 h at 220 rpm. The OD600 of the mixture was recorded after a 6-h subculture. The control group used 50 mmol.l-1 Na2HPO4-Na2HPO4 filter-sterilized buffer (pH 6.2) instead of lysozyme. Antibacterial activity of the purified lysozyme to S. aureus was assessed using the antibacterial ratio. Antibacterial ratio was calculated using the following equation of R = (A - B) ÷ A × 100%; in which, R was the antibacterial ratio (%); A was the OD600 value of the control group; and B was the OD600 value of the experimental group. The antibacterial effect curve was plotted with the antibacterial ratio on the y-axis and the logarithmic of the purified lysozyme concentration on the x-axis. The IC50 was defined as the purified lysozyme concentration, which resulted in a 50% decrease in the level of the antibacterial ratio, compared with untreated groups after a 6-h treatment. The IC50 value was calculated using GraphPad Prism software. The cell morphology of S. aureus after a 6-h lysozyme treatment (IC50 concentration) was reported using transmission electron microscopy (TEM). 2.5. Effects of heat treatment on anti-Staphylococcus aureus activity of the purified egg-white lysozyme To assess the anti-S. aureus effects of the heat-treated egg-white lysozyme, a specific concentration of the purified lysozyme solution was incubated at various temperatures for a set duration before assessing its anti-S. aureus activity using water bath. In this study, three key parameters were primarily investigated, including heat treatment temperature, time and initial lysozyme concentration. The temperatures were set at 37, 50, 60, 70, 80, 90 and 100 °C, respectively. The treatment times were set at 5, 10, 15, 60, 120 and 240 min, respectively. The initial lysozyme concentration were set at 601.6, 1000, 2000, 4000 and 6000 μg.ml-1, respectively. The three highlighted factors were optimized using one-factor-at-a-time method. After heat treatment, equal volumes of the lysozyme solution and S. aureus suspension were thoroughly mixed and co-cultured at 37 °C for 6 h at 220 rpm. The antibacterial ratio was then quantified. Untreated lysozyme was used as control group. To assess the effect of heat treatment on lysozyme molecular structure, circular dichroism (CD) spectroscopy was used to analyze changes in its secondary structural components before and after thermal exposure. The CD spectra were assessed using Jasco J-1500 spectropolarimeter (Jasco, Japan) with a 1-mm cell in the far-UV region from 190 to 300 nm. The concentration of lysozyme was 0.1 mg.ml-1 in 5 mmol.l-1 phosphate buffer (pH 6.2). 2.6. Inhibitory effects of phenolic acids combined with heat-treated egg-white lysozyme on Staphylococcus aureus 2.6.1. Compatibility screening of various phenolic acids with egg-white lysozyme In this experiment, the inhibitory effects of egg-white lysozyme combined with three phenolic acids (ferulic acid, caffeic acid and gallic acid) at various concentrations on S. aureus growth were assessed, respectively. The final concentrations of each phenolic acid were 200, 400, 800 and 1000 μg.ml-1, while the final concentration of egg-white lysozyme was set at 300.8 μg.ml-1. The anti-S. aureus activity analysis and antibacterial ratio calculation methods were based on those described in Section 1.4. For the control group, phenolic acids or lysozyme were replaced with 50 mmol.l-1 Na2HPO4-Na2HPO4 buffer solution (pH 6.2). 2.6.2. Effects of the heat-treated lysozyme and ferulic acid combination on Staphylococcus aureus growth The method for assessing the growth curves of S. aureus was based on Section 1.4. The heat treatment procedure for egg-white lysozyme was carried out according to Section 1.5, with the final concentration of heat-treated lysozyme adjusted to 300.8 μg.ml-1. For the control group, ferulic acid or lysozyme were substituted with 50 mmol.l-1 Na2HPO4-Na2HPO4 buffer solution (pH 6.2). 2.7. Numberized subsection inhibitory effects of Mycobacterium smegmatis acyltransferase combined with heat-treated egg-white lysozyme on Staphylococcus aureus growth The mechanism by which, M. smegmatis acyltransferase (MsAcT) combined with heat-treated egg white lysozyme inhibited the growth of S. aureus is illustrated in Fig. 6.1. The preparation of the purified MsAcT was based on the methods described by Jia et al. [15]. Briefly, recombinant Escherichia coli BL21(DE3) strain was inoculated to Luria-Bertani (LB) broth and grown at 30 °C. The IPTG was added to culture broth to the final concentration of 1 mmol.l-1, when the OD600 reached 0.6–0.8. After 14 h interval, cell pellet was collected, resuspended using loading buffer (20 mmol.l-1 pH 7.4 Na2HPO4-NaH2PO4, 20 mmol.l-1 imidazole and 500 mmol.l-1 NaCl) and then lysed using sonication. The supernatant from the cell lysate was collected and directly loaded on the HisTrap HP affinity chromatography column pre-equilibrated with loading buffer, respectively. Recombinant protein was eluted with a linear gradient of 20 ml of 20–500 mmol.l-1 imidazole in the buffer with a flow rate of 0.8 ml.min-1. The fractions with pure MsAcT were pooled and dialyzed against 20 mmol.l-1 Na2HPO4-NaH2PO4 (pH 6.2) buffer overnight at 4 ◦C. The hydrolysis activity of MsAcT to NAG was assessed according to Jiang et al. [16] and Muzzarelli and Rocchetti [17]. In brief, the reaction mixture contained 20 μg.ml-1 NAG in 20 mmol.l-1 Na2HPO4-Na2HPO4 buffer (pH 6.2). Appropriately diluted MsAcT was added into the reaction mixture to create a linear dependence of the reaction rate to protein concentration. The reaction was carried out at 37 ◦C and the kinetics was detected for 3 h at 202 nm. The specific procedure for inhibiting S. aureus growth using MsAcT combined with heat-treated egg-white lysozyme was carried out as follows: MsAcT, heat-treated egg white lysozyme and buffer solutions were sterilized using 0.22-μm filters. Then, 200 μl of appropriately diluted log-phase S. aureus cells were mixed with 200 μl of 1 mg.ml-1 MsAcT solution, while the control group received 200 μl of 50 mmol.l-1 NaH2PO4-Na2HPO4 solution (pH 6.2). After incubation at 37 °C for 3 h, 200 μl of the heat-treated egg-white lysozyme (final concentration of 300.8 μg.ml-1) were added to the experimental and control groups and mixed thoroughly, followed by incubation at 37 °C for 3 h. The samples were then centrifuged at 12,000 rpm for 3 min to separate the supernatant and the bacterial cell pellet. The protein concentration in the supernatant was measured to calculate the increase in protein, while the number of viable bacteria in the pellet was assessed according to the National Food Safety Standard of China (GB4789.2-2022, Food Microbiological Examination: Determination of Total Bacterial Count). Results and Discussion 3.1. Purification and characterization of egg-white lysozyme The purification of egg-white lysozyme was achieved through ammonium sulfate precipitation and CM-Sepharose fast flow column chromatography, resulting in 8.7-fold purification and yield of 41.0% (Table 1). Cation exchange chromatography verified particularly effective, accounting for 5.8-fold increase in specific activity. The purified enzyme was verified as homogenous using SDS-PAGE, showing a single band at 14.3 kDa (Fig. 1, Lane 6), which was similar to the molecular mass of egg-white lysozyme [18]. Its specific activity was assessed as 27,407.4 U.mg-1 using M. lysodeikticus cells as substrate. This value was less than that reported by Chen et al. [18], a discrepancy likely attributable to the use of whole cells in this assay instead of the isolated cell walls used in the highlighted study. A gradual loss of total activity was observed throughout purification. Particularly, lysozyme was detected in pellets at a relatively low ammonium sulfate saturation (40%), suggesting the potential formation of insoluble aggregates under these conditions, as previously documented [19, 20]. For practical uses, it is critical to state that the growth state of M. lysodeikticus, affected by culture conditions and equipment, significantly affects the assessed specific activity [11, 13, 21]. Therefore, standardizing the substrate by setting the growth curve and harvesting log-phase cells under local laboratory conditions is essential for accurate activity assessment. In this study, the buffer solution for the assessment of the egg-white lysozyme activity according to the National Standard of the People's Republic of China (GB/T 30990-2014) requirements included 50 mmol.l-1 Na2HPO4-NaH2PO4 buffer (pH 6.2). In practical fields, particularly in the food processing industry, buffer solutions with pH 6.2 are rarely used. Therefore, it is essential to assess optimal pH and temperature under the specific conditions necessary for the target uses, when assessing using effectiveness of egg-white lysozyme. 3.2. Anti-Staphylococcus aureus activity and mechanism of the purified egg-white lysozyme The growth curve of S. aureus was characterized, identifying a log phase from 1 to 8 h (Fig. 2A), similar to the previous reports [22]. Cells from the exponential phase (2 h) were used for the assays. The IC50 of native lysozyme against S. aureus was 300.8 μg.ml-1 (Fig. 2B). The antibacterial efficacy of lysozyme is affected by the composition and sequence of bacterial cell wall peptidoglycan, as well as the physiological state of the enzyme [23, 24]. Lysozyme fights microbes through bacteriostatic, bactericidal and bacteriolytic mechanisms [25]. The transmission electron microscopy (TEM) images provided direct evidence of the bacteriolytic action, showing damaged S. aureus cell walls with distinct light/dark contrast and the collapse of cells, leading to leakage of intracellular contents (Fig. 2C, arrows). Despite this significant effect, the inhibitory activity of the native lysozyme is difficult to sustain over extended times (Figs. 2A, 5A), highlighting a limitation for its use as a standalone antimicrobial agent. 3.3. Enhancement of anti-Staphylococcus aureus activity using heat treatment and structural changes Heat treatment under optimized conditions (600 μg.ml-1, 90°C, 15 min, pH 6.2) enhanced the anti-S. aureus activity of lysozyme by 35.1%, achieving 82.5% inhibition compared to the native enzyme (Fig. 3A–C). This increase in activity after thermal denaturation was similar to that against other microbes such as SARS-CoV-2 and Bacillus subtilis [26, 27, 28]. Structural analysis revealed the reason behind this enhancement as a significant rearrangement of secondary structure occurred, with α-helix content decreasing from 35.59 to 23.60% and β-sheet, β-turn and random coil structures increasing (Fig. 3D, Table 2). This unfolding and proliferation of β-sheets drived the formation of fibrillar aggregates [30,29], which were postulated to perforate microbial membranes, a mechanism distinct from the native enzyme peptidoglycan hydrolysis [27]. This suggested that structural modification wa a viable strategy to improve the efficacy of lysozyme. 3.4. Synergistic anti-Staphylococcus aureus effects of heat-treated egg-white lysozyme and phenolic acids From the phenolic acids (ferulic, caffeic and gallic acids), ferulic acid showed the highest inhibition (52.2%) at 200 μg.ml-1, though differences diminished at higher concentrations (1000 μg.ml-1), where all acids reached ~99% inhibition (Fig. 4A–C). Ferulic acid is reported to inhibit S. aureus by suppressing tetK and MsrA efflux pumps on the bacterial membrane [31,32]. A combination of 400 μg.ml-1 ferulic acid with native lysozyme (300.8 μg.ml-1) showed a synergistic effect, increasing inhibition by respectively 12.3 and 29%, compared to either compound alone (Fig. 4A). This synergy was further increased, when ferulic acid was combined with heat-treated lysozyme, reducing bacterial biomass (OD600) by additional 18.7%, compared to the combination with native enzyme (Fig. 5A). The optimal protocol involved sequential addition of heat-treated lysozyme added at time zero, followed by ferulic acid after 6 h. This not only delayed the entry into the log phase by an additional hour but also decreased final biomass by 48.8% (Fig. 5B). This demonstrated that combining lysozyme with other antibacterial compounds, particularly after structural modification, could significantly enhance and prolong its inhibitory effect, potentially broadening its antibacterial spectrum [12,33]. However, when designing such combinations, concentration and addition sequence of egg-white lysozyme and phenolic acid, biocompatibility, reaction condition and differences in the mechanisms of action must carefully be addressed. Otherwise, adverse effects may occur. For e