Plasmids for Optimizing Expression of Recombinant Proteins in <em>E. coli</em>

Plasmids are important vectors for the transfer of genetic material among microbes. The transfer of plasmids causes transmission of genes involved in pathogenesis and survival, to the host bacteria leading to their evolution and adaptation to diverse environmental conditions. A large number of plasmids of varying sizes have been discovered and isolated from various microorganisms. Plasmids are also valuable tools to genetically manipulate microbes for various purposes including production of recombinant proteins. Escherichia coli is the most preferred microbe for production of recombinant proteins, due to rapid growth rate, cost-effectiveness, high yield of the recombinant proteins and easy scale-up process. Several plasmids have been designed to optimize the expression of heterologous proteins in E. coli. In order to circumvent the issues of protein refolding, the codon usage in E. coli, the absence of post-translational modifications, such as glycosylation and low recovery of functionally active recombinant proteins, various plasmids have been designed and constructed. This chapter summarizes the recent technological advancements that have extended the use of the E. coli expression system to produce more complex proteins, including glycosylated recombinant proteins and therapeutic antibodies

A Sensitive and Rapid Assay for Investigating Vertical Transmission of Hepatitis B Virus via Male Germ Line Using EGFP Vector as Reporter

Hepatitis B virus (HBV) constitutes a serious menace to man. DNA recombination and sequencing, interspecific in vitro fertilization, single-embryo PCR and RT-PCR were employed to establish a sensitive and rapid assay for exploring the vertical transmission of viruses via male germ line. Plasmid pIRES2-EGFP-HBs which expressed enhanced green fluorescent protein as reporter for the expression of hepatitis B virus S gene was successfully constructed and confirmed by PCR, EcoR I and Sal I digestion, and DNA sequencing. After exposure to the plasmid, human spermatozoa were used to fertilize with zona-free hamster ova. Two-cell embryos were collected and classified into group A with green fluorescence and group B without green fluorescence under fluorescence microscope. The results showed that HBs DNA positive bands were detected in the embryos with green fluorescence (PCR and RT-PCR) and positive control (PCR) indicating expression of pIRES2-EGFP-HBs, and not observed in the embryos without green fluorescence and negative controls (PCR and RT-PCR) indicating no pIRES2-EGFP-HBs in the cells. The advantages and application foreground of this assay for study on vertical transmission of viruses such as HCV, HIV, HPV, and SARS via germ line were discussed

Role of static fluid MR urography in detecting post urinary diversion complications

Aim of work: The aim of the study was to assess the diagnostic performance of static MR urography in detection of post cystectomy complications &amp; the ability of static fluid MR urography in visualization of urinary tract segments.Material &amp; methods: We prospectively reviewed 21 MR urograms with urinary diversion. The most common surgical procedures included Ileal conduit &amp; Ileocecal neobladder diversion.Material &amp; methods: Magnetic resonance urography examinations were performed with 1.5-T MR scanners. T2 weighted (static fluid) MR urography techniques were done, in addition to conventional T1- and T2-weighted axial and coronal sequences. Urinary tract was divided in different parts: pelvicalyceal systems, upper, mid and lower ureteric segments &amp; the reservoir or conduit Imaging features of the urinary collecting systems were evaluated for their visualization and complications detection.Results: T2-weighted MR urography could demonstrate 95% of urinary tract segments &amp; together with conventional MR sequences all urinary tract segments can be visualized. Urinary diversion related complications were encountered included in 15 patients (71.4%) &amp; no urological complications were seen in 6 patients (28.6%).Conclusion: Comprehensive T2-weighted MR urography is an effective imaging method for the visualization of the urinary system and detection of early and late postoperative complications in patients with urinary diversion.Keywords: MR urography, Urinary diversion, Cancer bladde

Comparative molecular studies of halophilic bacteria from saline water and soil in the Saudi environment

Halophilic bacteria are a microorganism that grows optimally in the presence of the very high concentration of sodium chloride. Halophiles are vital sources of various enzymes including hydrolases, which are very stable and catalytically highly efficient at high salt concentration and other extreme conditions such as high temperature, pH and presence of organic solvents.  Several hydrolases such as amylases, proteases, and lipases have been obtained from halophilic bacteria and are commonly used for various industrial applications. We initiated a screening project to isolate and characterize the halophilic bacteria from the Red Sea, which is one of the saltiest bodies of water in the world. Water and soil samples, collected from the Red Sea coast, Jeddah, Saudi Arabia, were screened for isolation of halophilic bacteria. Ten bacterial isolates were obtained, which were characterized by biochemical tests and 16S rRNA gene sequencing. Hydrolase producing bacteria among the isolates were screened by plate assay on starch and gelatin agar plates for amylase and protease, respectively.  Two bacterial isolates i.e Bacillus haynesii and Enterobacter cloacae subsp. were found to possess significant amylase and protease activity. Further characterization of both the strains is in progress

Assessing Drought Tolerance of Newly Developed Tissue-Cultured Canola Genotypes under Varying Irrigation Regimes

This article belongs to the Special Issue Crop Tolerance under Biotic and Abiotic Stresses[Abstract] Drought is a major abiotic stress that greatly affects canola growth, production, and quality. Moreover, water scarcity is projected to be more severe and frequent as a result of climate change, in particular in arid environments. Thereupon, developing drought-tolerant and high-yielding canola genotypes has become more critical to sustaining its production and ensuring global food security with the continuing population growth. In the present study, ten canola genotypes comprising six developed tissue-cultured canola genotypes, two exotic genotypes, and two commercial cultivars were evaluated under four irrigation regimes. The applied irrigation regimes were well-watered (100% crop evapotranspiration, ETc), mild drought (80% ETc), moderate drought (60% ETc), and severe drought (40% ETc) conditions. Drought-stress treatments (80, 60, and 40% ETc) gradually reduced the chlorophyll content, relative water content, flowering time, days to maturity, plant height, number of pods, number of branches, seed yield, and oil percentage, and increased proline, phenolic, anthocyanin, and glycine betaine contents. The evaluated genotypes exhibited varied responses to drought-stress conditions. The developed tissue-cultured genotypes T2, T3, and T1, as well as exotic genotype Torpe, possessed the highest performance in all evaluated parameters and surpassed the other tested genotypes under water-deficit conditions. Overall, our findings elicited the superiority of certain newly developed tissue-cultured genotypes and exotic ones compared with commercial cultivars, which could be exploited in canola breeding under water-deficit conditions.This research was funded by the Researchers Supporting Project number (RSPD-2023R730), King Saud University, Riyadh, Saudi ArabiaKing Saud University (Riad, Arabia Saudí); RSPD-2023R73

Hepatobiliary manifestations following two-stages elective laparoscopic restorative proctocolectomy for patients with ulcerative colitis: A prospective observational study

BACKGROUNDHepatobiliary manifestations occur in ulcerative colitis (UC) patients. The effect of laparoscopic restorative proctocolectomy (LRP) with ileal pouch anal anastomosis (IPAA) on hepatobiliary manifestations is debated.AIMTo evaluate hepatobiliary changes after two-stages elective laparoscopic restorative proctocolectomy for patients with UC.METHODSBetween June 2013 and June 2018, 167 patients with hepatobiliary symptoms underwent two-stage elective LRP for UC in a prospective observational study. Patients with UC and having at least one hepatobiliary manifestation who underwent LRP with IPAA were included in the study. The patients were followed up for four years to assess the outcomes of hepatobiliary manifestations.RESULTSThe patients' mean age was 36 +/- 8 years, and males predominated (67.1%). The most common hepatobiliary diagnostic method was liver biopsy (85.6%), followed by Magnetic resonance cholangiopancreatography (63.5%), Antineutrophil cytoplasmic antibodies (62.5%), abdominal ultrasonography (35.9%), and Endoscopic retrograde cholangiopancreatography (6%). The most common hepatobiliary symptom was Primary sclerosing cholangitis (PSC) (62.3%), followed by fatty liver (16.8%) and gallbladder stone (10.2%). 66.4% of patients showed a stable course after surgery. Progressive or regressive courses occurred in 16.8% of each. Mortality was 6%, and recurrence or progression of symptoms required surgery for 15%. Most PSC patients (87.5%) had a stable course, and only 12.5% became worse. Two-thirds (64.3%) of fatty liver patients showed a regressive course, while one-third (35.7%) showed a stable course. Survival rates were 98.8%, 97%, 95.8%, and 94% at 12 mo, 24 mo, 36 mo, and at the end of the follow-up.CONCLUSIONIn patients with UC who had LRP, there is a positive impact on hepatobiliary disease. It caused an improvement in PSC and fatty liver disease. The most prevalent unchanged course was PSC, while the most common improvement was fatty liver disease

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10&nbsp;years; 78.2% included were male with a median age of 37&nbsp;years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

The effect of type of femoral component fixation on mortality and morbidity after hip hemiarthroplasty:A systematic review and meta-analysis

Background: Hip hemiarthroplasty is a well-established treatment of displaced femoral neck fracture, although debate exists over whether cemented or uncemented fixation is superior. Uncemented prostheses have typically been used in younger, healthier patients and cemented prostheses in older patients with less-stable bone. Also, earlier research has suggested that bone cement has cytotoxic effects and may trigger cardiovascular and respiratory adverse events. Questions/Purposes: The aim of this systematic review and meta-analysis was to compare morbidity and mortality rates after cemented and uncemented hemiarthroplasty for the treatment of displaced femoral neck fractures in elderly patients. Methods: Using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, we searched seven medical databases for randomized clinical trials and observational studies. We compared cemented and uncemented hemiarthroplasty using the Harris Hip Score (HHS), as well as measures of postoperative pain, mortality, and complications. Data were extracted and pooled as risk ratios or standardized mean difference with their corresponding 95% confidence intervals in a meta-analysis model. Results: The meta-analysis included 34 studies (12 randomized trials and 22 observational studies), with a total of 42,411 patients. In the pooled estimate, cemented hemiarthroplasty was associated with less risk of postoperative pain than uncemented hemiarthroplasty. There were no significant differences between groups regarding HHS or rates of postoperative mortality, pulmonary embolism, cardiac arrest, myocardial infarction, acute cardiac arrhythmia, or deep venous thrombosis. Conclusions: While we found that cemented hemiarthroplasty results in less postoperative pain than uncemented hemiarthroplasty in older patients with femoral neck fracture, the lack of significant differences in functional hip scores, mortality, and complications was surprising. Further high-level research is needed

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P &lt; 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely