Intracellular \u3cem\u3eSalmonella\u3c/em\u3e delivery of an exogenous immunization antigen refocuses CD8 T cells against cancer cells, eliminates pancreatic tumors and forms antitumor immunity

Introduction: Immunotherapies have shown great promise, but are not effective for all tumors types and are effective in less than 3% of patients with pancreatic ductal adenocarcinomas (PDAC). To make an immune treatment that is effective for more cancer patients and those with PDAC specifically, we genetically engineered Salmonella to deliver exogenous antigens directly into the cytoplasm of tumor cells. We hypothesized that intracellular delivery of an exogenous immunization antigen would activate antigen-specific CD8 T cells and reduce tumors in immunized mice. Methods: To test this hypothesis, we administered intracellular delivering (ID) Salmonella that deliver ovalbumin as a model antigen into tumor-bearing, ovalbumin-vaccinated mice. ID Salmonella delivers antigens by autonomously lysing in cells after the induction of cell invasion. Results: We showed that the delivered ovalbumin disperses throughout the cytoplasm of cells in culture and in tumors. This delivery into the cytoplasm is essential for antigen cross-presentation. We showed that co-culture of ovalbumin-recipient cancer cells with ovalbumin-specific CD8 T cells triggered a cytotoxic T cell response. After the adoptive transfer of OT-I CD8 T cells, intracellular delivery of ovalbumin reduced tumor growth and eliminated tumors. This effect was dependent on the presence of the ovalbumin-specific T cells. Following vaccination with the exogenous antigen in mice, intracellular delivery of the antigen cleared 43% of established KPC pancreatic tumors, increased survival, and prevented tumor re-implantation. Discussion: This response in the immunosuppressive KPC model demonstrates the potential to treat tumors that do not respond to checkpoint inhibitors, and the response to re-challenge indicates that new immunity was established against intrinsic tumor antigens. In the clinic, ID Salmonella could be used to deliver a protein antigen from a childhood immunization to refocus pre-existing T cell immunity against tumors. As an off-the-shelf immunotherapy, this bacterial system has the potential to be effective in a broad range of cancer patients

Intracellular delivery of protein drugs with an autonomously lysing bacterial system reduces tumor growth and metastases

Critical cancer pathways often cannot be targeted because of limited efficiency crossing cell membranes. Here we report the development of a Salmonella-based intracellular delivery system to address this challenge. We engineer genetic circuits that (1) activate the regulator flhDC to drive invasion and (2) induce lysis to release proteins into tumor cells. Released protein drugs diffuse from Salmonella containing vacuoles into the cellular cytoplasm where they interact with their therapeutic targets. Control of invasion with flhDC increases delivery over 500 times. The autonomous triggering of lysis after invasion makes the platform self-limiting and prevents drug release in healthy organs. Bacterial delivery of constitutively active caspase-3 blocks the growth of hepatocellular carcinoma and lung metastases, and increases survival in mice. This success in targeted killing of cancer cells provides critical evidence that this approach will be applicable to a wide range of protein drugs for the treatment of solid tumors

Multiparent-Derived, Marker-Assisted Introgression Lines of the Elite Indian Rice Cultivar, ‘Krishna Hamsa’ Show Resistance against Bacterial Blight and Blast and Tolerance to Drought

Major biotic stresses viz., bacterial blight (BB) and blast and brown plant hopper (BPH) coupled with abiotic stresses like drought stress, significantly affect rice yields. To address this, marker-assisted intercross (IC) breeding involving multiple donors was used to combine three BB resistance genes—xa5, xa13 and Xa21, two blast resistance genes—Pi9 and Pi54, two BPH resistance genes—Bph20 and Bph21, and four drought tolerant quantitative trait loci (QTL)—qDTY1.1, qDTY2.1, qDTY3.1 and qDTY12.1—in the genetic background of the elite Indian rice cultivar ‘Krishna Hamsa’. Three cycles of selective intercrossing followed by selfing coupled with foreground selection and phenotyping for the target traits resulted in the development of 196 introgression lines (ILs) with a myriad of gene/QTL combinations. Based on the phenotypic reaction, the ILs were classified into seven phenotypic classes of resistance/tolerance to the following: (1) BB, blast and drought—5 ILs; (2) BB and blast—10 ILs; (3) BB and drought—9 ILs; (4) blast and drought—42 ILs; (5) BB—3 ILs; (6) blast—84 ILs; and (7) drought—43 ILs; none of the ILs were resistant to BPH. Positive phenotypic response (resistance) was observed to both BB and blast in 2 ILs, BB in 9 ILs and blast in 64 ILs despite the absence of corresponding R genes. Inheritance of resistance to BB and/or blast in such ILs could be due to the unknown genes from other parents used in the breeding scheme. Negative phenotypic response (susceptibility) was observed in 67 ILs possessing BB-R genes, 9 ILs with blast-R genes and 9 ILs harboring QTLs for drought tolerance. Complex genic interactions and recombination events due to the involvement of multiple donors explain susceptibility in some of the marker positive ILs. The present investigation successfully demonstrates the possibility of rapid development of multiple stress-tolerant/resistant ILs in the elite cultivar background involving multiple donors through selective intercrossing and stringent phenotyping. The 196 ILs in seven phenotypic classes with myriad of gene/QTL combinations will serve as a useful genetic resource in combining multiple biotic and abiotic stress resistance in future breeding programs

Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Background: In an era of shifting global agendas and expanded emphasis on non-communicable diseases and injuries along with communicable diseases, sound evidence on trends by cause at the national level is essential. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) provides a systematic scientific assessment of published, publicly available, and contributed data on incidence, prevalence, and mortality for a mutually exclusive and collectively exhaustive list of diseases and injuries. Methods: GBD estimates incidence, prevalence, mortality, years of life lost (YLLs), years lived with disability (YLDs), and disability-adjusted life-years (DALYs) due to 369 diseases and injuries, for two sexes, and for 204 countries and territories. Input data were extracted from censuses, household surveys, civil registration and vital statistics, disease registries, health service use, air pollution monitors, satellite imaging, disease notifications, and other sources. Cause-specific death rates and cause fractions were calculated using the Cause of Death Ensemble model and spatiotemporal Gaussian process regression. Cause-specific deaths were adjusted to match the total all-cause deaths calculated as part of the GBD population, fertility, and mortality estimates. Deaths were multiplied by standard life expectancy at each age to calculate YLLs. A Bayesian meta-regression modelling tool, DisMod-MR 2.1, was used to ensure consistency between incidence, prevalence, remission, excess mortality, and cause-specific mortality for most causes. Prevalence estimates were multiplied by disability weights for mutually exclusive sequelae of diseases and injuries to calculate YLDs. We considered results in the context of the Socio-demographic Index (SDI), a composite indicator of income per capita, years of schooling, and fertility rate in females younger than 25 years. Uncertainty intervals (UIs) were generated for every metric using the 25th and 975th ordered 1000 draw values of the posterior distribution. Findings: Global health has steadily improved over the past 30 years as measured by age-standardised DALY rates. After taking into account population growth and ageing, the absolute number of DALYs has remained stable. Since 2010, the pace of decline in global age-standardised DALY rates has accelerated in age groups younger than 50 years compared with the 1990–2010 time period, with the greatest annualised rate of decline occurring in the 0–9-year age group. Six infectious diseases were among the top ten causes of DALYs in children younger than 10 years in 2019: lower respiratory infections (ranked second), diarrhoeal diseases (third), malaria (fifth), meningitis (sixth), whooping cough (ninth), and sexually transmitted infections (which, in this age group, is fully accounted for by congenital syphilis; ranked tenth). In adolescents aged 10–24 years, three injury causes were among the top causes of DALYs: road injuries (ranked first), self-harm (third), and interpersonal violence (fifth). Five of the causes that were in the top ten for ages 10–24 years were also in the top ten in the 25–49-year age group: road injuries (ranked first), HIV/AIDS (second), low back pain (fourth), headache disorders (fifth), and depressive disorders (sixth). In 2019, ischaemic heart disease and stroke were the top-ranked causes of DALYs in both the 50–74-year and 75-years-and-older age groups. Since 1990, there has been a marked shift towards a greater proportion of burden due to YLDs from non-communicable diseases and injuries. In 2019, there were 11 countries where non-communicable disease and injury YLDs constituted more than half of all disease burden. Decreases in age-standardised DALY rates have accelerated over the past decade in countries at the lower end of the SDI range, while improvements have started to stagnate or even reverse in countries with higher SDI. Interpretation: As disability becomes an increasingly large component of disease burden and a larger component of health expenditure, greater research and developm nt investment is needed to identify new, more effective intervention strategies. With a rapidly ageing global population, the demands on health services to deal with disabling outcomes, which increase with age, will require policy makers to anticipate these changes. The mix of universal and more geographically specific influences on health reinforces the need for regular reporting on population health in detail and by underlying cause to help decision makers to identify success stories of disease control to emulate, as well as opportunities to improve. Funding: Bill & Melinda Gates Foundation. © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licens

CREATION OF A TUMOR SPECIFIC, SALMONELLA BASED, INTRACELLULAR CANCER THERAPY

Sixty percent of all proteins are located inside cells. Many of these proteins are involved in pathways that regulate a variety of critical cancer cell survival and immunomodulatory processes. However, conventional macromolecular therapies targeting these intracellular pathways in cancer face several transport barriers including, tumor selective accumulation, dispersion, cell internalization and endosomal release. Therefore, an effective delivery vehicle is needed to circumvent the transport limitations associated with macromolecular therapies. Salmonella is an ideal intracellular macromolecular delivery vehicle for cancer therapy/immunotherapy. Non-pathogenic versions of the bacteria colonize and grow in tumors at ratios greater than ten thousand to one over any other organ. The bacteria are highly motile, disperse and efficiently invade non-phagocytic, epithelial cells. After cell invasion, Salmonella activate a unique set of genes selectively inside cells to upregulate type three secretion system-two activity, which, enables intracellular survival. This combination of traits is unique to Salmonella, making genetically engineered versions of the bacteria ideal for intracellular therapeutic delivery selectively within tumor cells. This thesis had two purposes. The first was to determine the critical driving mechanisms governing intracellular therapeutic delivery in tumor cells and genetically engineer a delivery strain of Salmonella based on this information. The second was to demonstrate, for the very first time, that the engineered Salmonella could deliver protein antigen into tumor cells and refocus preexisting, vaccine induced, immune cells to target cancer. We hypothesized that controlled expression of the master motility regulator, flhDC, in Salmonella drives tumor colonization, bacterial dispersion, invasion and protein delivery selectively inside tumor cells. To test this hypothesis, we employed a range of genetic engineering techniques, cell-based infection assays, in vitro tumor-on-a-chip and in vivo infection/tumor models to elucidate the driving delivery mechanisms of engineered Salmonella. Controlled expression of flhDC in engineered Salmonella enabled high levels of intracellular protein delivery selectively inside tumor cells. Once the delivery strain was created and optimized, we hypothesized that the engineered Salmonella could deliver the model vaccine antigen, ovalbumin, into tumor cells and refocus preexisting, vaccine induced immune cells to attack cancer. To test this hypothesis, we employed cell-based, syngeneic, and transgenic mouse infection/tumor models. Delivering the model vaccine antigen, ovalbumin, into tumors with engineered Salmonella refocused vaccine associated immune cells, including ovalbumin specific, CD8 T cells to combat and cure cancer in a subset of mice. This result demonstrates that Salmonella engineered to deliver vaccine antigen into tumors could be employed as an effective, off-the-shelf cancer immunotherapy with broad applicability in previously vaccinated cancer patients

Arthrodesis with the Ilizarov ring fixator for severe ankle arthritis

Introduction End-stage ankle arthritis is a very painful and disabling pathology, associated with deformity. Infection, poor skin condition, chronic smoking, Charcot arthropathy may not only affect selection of treatment method but also union, leading to unfortunate amputation. Ankle arthrodesis is indicated in advanced ankle arthritis. A variety of fixation methods are available for arthrodesis ranging from internal to external fixation. The Ilizarov ring fixator is a dynamic versatile fixation method. It is a biomechanically stable and minimally invasive method which promotes bone union and has advantage of initiating early weight-bearing and simultaneous deformity correction. We describe our experience in Ilizarov ring fixator application for ankle arthrodesis in 5 patients with severe ankle arthritis and their functional outcome. Materials and Methods This retrospective study was conducted in 5 ankle arthrodesis cases using the Ilizarov ring fixator application from July 2021 to October 2022 in the department of orthopaedics, Jaipur national university, India. Average age of patient was 52 years (range, 40-65). Among included patients one patient had chronic osteomyelitis of the distal tibia and severe arthrosis of the ankle joint with a non-healing ulcer, two patients had post-traumatic arthrosis following talus and distal tibia plafond fracture, Charcot ankle arthropathy and tuberculosis of the ankle joint was detected in two patients respectively. Postoperative pain relief, deformity correction and radiological union at the fusion site were defined as success. Results Fusion was achieved in all patients (100%). Early post-operative ambulation and full weight-bearing was initiated in every case. Pin-tract infection was the commonest complication. Shortening due to arthrodesis was less than 2.5 cm so limb lengthening was not done. Frame removal time was 12 to 14 weeks (average time, 13 weeks). Visual analogue scale was used in all cases. It was in the range of 2 to 3 points preoperatively and 7 to 9 post-operatively after arthrodesis. Average follow-up period was 6 months and it is still underway. AOFAS score was used for functional assessment. Conclusion Ilizarov ring fixator application can be considered as versatile, biomechanically stable, minimally invasive method for ankle arthrodesis in severe ankle arthritis associated with poor soft tissue condition, posttraumatic arthritis, infection, deformity, bone loss, Charcot arthropathy

Weapon Detection in Surveillance Videos Using YOLOV8 and PELSF-DCNN

Weapon detection (WD) provides early detection of potentially violent situations. Despite deep learning (DL) algorithms and sophisticated closed-circuit television (CCTVs), detecting weapons is still a difficult task. So, this paper proposes a WD model using PELSF-DCNN. Initially, the input video is converted into frames and pre-processed. The objects in the pre-processed frames are detected using the YOLOv8. In meantime, motion estimation is done using the DS algorithm in the pre-processed images to cover all the information. Then, the detected weapons undergo a sliding window process by considering the motion estimated frames. The silhouette score is calculated for detected humans and other objects. Now, the features are extracted and the important features are selected using the CSBO algorithm. The selected features and the output of YOLOv8 are given to the PELSF-DCNN classifier. Finally, the confidence score is calculated for the frame to define the number of weapons. In an experimental evaluation, the proposed method is found to be more efficient than the existing methods

The motility regulator flhDC drives intracellular accumulation and tumor colonization of Salmonella

Abstract Background Salmonella have potential as anticancer therapeutic because of their innate tumor specificity. In clinical studies, this specificity has been hampered by heterogeneous responses. Understanding the mechanisms that control tumor colonization would enable the design of more robust therapeutic strains. Two mechanisms that could affect tumor colonization are intracellular accumulation and intratumoral motility. Both of these mechanisms have elements that are controlled by the master motility regulator flhDC. We hypothesized that 1) overexpressing flhDC in Salmonella increases intracellular bacterial accumulation in tumor cell masses, and 2) intracellular accumulation of Salmonella drives tumor colonization in vitro. Methods To test these hypotheses, we transformed Salmonella with genetic circuits that induce flhDC and express green fluorescent protein after intracellular invasion. The genetically modified Salmonella was perfused into an in vitro tumor-on-a-chip device. Time-lapse fluorescence microscopy was used to quantify intracellular and colonization dynamics within tumor masses. A mathematical model was used to determine how these mechanisms are related to each other. Results Overexpression of flhDC increased intracellular accumulation and tumor colonization 2.5 and 5 times more than control Salmonella, respectively (P < 0.05). Non-motile Salmonella accumulated in cancer cells 26 times less than controls (P < 0.001). Minimally invasive, ΔsipB, Salmonella colonized tumor masses 2.5 times less than controls (P < 0.05). When flhDC was selectively induced after penetration into tumor masses, Salmonella both accumulated intracellularly and colonized tumor masses 2 times more than controls (P < 0.05). Mathematical modeling of tumor colonization dynamics demonstrated that intracellular accumulation increased retention of Salmonella in tumors by effectively causing the bacteria to bind to cancer cells and preventing leakage out of the tumors. These results demonstrated that increasing intracellular bacterial density increased overall tumor colonization and that flhDC could be used to control both. Conclusions This study demonstrates a mechanistic link between motility, intracellular accumulation and tumor colonization. Based on our results, we envision that therapeutic strains of Salmonella could use inducible flhDC to drive tumor colonization. More intratumoral bacteria would enable delivery of higher therapeutic payloads into tumors and would improve treatment efficacy

A practicable approach for periodontal classification

The Diagnosis and classification of periodontal diseases has remained a dilemma since long. Two distinct concepts have been used to define diseases: Essentialism and Nominalism. Essentialistic concept implies the real existence of disease whereas; nominalistic concept states that the names of diseases are the convenient way of stating concisely the endpoint of a diagnostic process. It generally advances from assessment of symptoms and signs toward knowledge of causation and gives a feasible option to name the disease for which etiology is either unknown or it is too complex to access in routine clinical practice. Various classifications have been proposed by the American Academy of Periodontology (AAP) in 1986, 1989 and 1999. The AAP 1999 classification is among the most widely used classification. But this classification also has demerits which provide impediment for its use in day to day practice. Hence a classification and diagnostic system is required which can help the clinician to access the patient′s need and provide a suitable treatment which is in harmony with the diagnosis for that particular case. Here is an attempt to propose a practicable classification and diagnostic system of periodontal diseases for better treatment outcome