Antigen Typing Research Articles

Investigating on sensing mechanism of MoS2-FET biosensors in response to proteins

Field-effect transistor (FET) biosensors based on two-dimensional materials have gained extensive attention due to their high sensitivity, label-free detection capability, and fast response. Molybdenum disulfide (MoS2), with tunable bandgap, high surface-to-volume ratio, and smooth surface without dangling bonds, is a promising material for FET biosensors. Previous reports have demonstrated the fabrication of MoS2-FET biosensors and their high sensitivity detection of proteins. However, most prior research has focused on the realization of MoS2-FETs for detecting different kinds of proteins or molecules, while comprehensive analysis of the sensing mechanism and dominant device factors of MoS2-FETs in response to proteins is yet to investigate. In this study, we first fabricated MoS2-FET biosensor and detected different types of proteins (immunoglobulin G (IgG), β-actin, and prostate-specific antigen (PSA)). Secondly, we built the model of the device and analyzed the sensing mechanism of MoS2-FETs in response to proteins. Experimental and modeling results showed that the induced doping effect and gating effect caused by the target protein binding to the device surface were the major influential factors. Specifically, the channel doping concentration and gate voltage (V g) offset exhibited monotonic change as the concentration of the protein solution increases. For example, the channel doping concentration increased up to ∼37.9% and the V g offset was ∼−1.3 V with 10−7 μg μl−1 IgG. The change was less affected by the device size. We also investigated the effects of proteins with opposite acid–base properties (β-actin and PSA) to IgG on the device sensing mechanism. β-actin and PSA exhibited behavior opposite to that of IgG. Additionally, we studied the response behavior of MoS2-FETs with different dimensions and dielectric materials (channel length, MoS2 thickness, dielectric layer thickness, dielectric layer material) to proteins. The underlying mechanisms were discussed in details. This study provides valuable guidelines for the design and application of MoS2-FET biosensors.

Structure-Based Multi-Targeted Molecular Docking and Dynamic Simulation of Soybean-Derived Isoflavone Genistin as a Potential Breast Cancer Signaling Proteins Inhibitor.

Globally, breast cancer (BC), the second-biggest cause of cancer death, occurs due to unregulated cell proliferation leading to metastasis to other parts of the human organ. Recently, the exploration of naturally derived anticancer agents has become popular due to their fewer adverse effects. Among the natural products, soybean is a very well-known legume that contains important bioactive compounds such as diadazine, glycetin, genistein, and genistin. Therefore, keeping its therapeutic potential in mind, multi-targeted molecular docking and simulation studies were conducted to explore the potential role of soybean-derived isoflavone genistin against several breast cancer-signaling proteins (ER-alpha, ER-Beta, collapsin response mediator protein 2, CA 15-3, human epidermal growth factor receptor 2). A comparative study of the genistin-protein docked complex was explored to investigate its potential role in BC. The molecular binding energy (∆G) of the docked complex was calculated along with ADMET properties. The molecular docking score of genistin with ubiquitin-like protein activation complex-a type of Cancer Antigen (CA) 15.3 (PDB ID-2NVU, 5T6P, and 1YX8) showed the highest binding energy, ranging from -9.5 to -7.0 Kcal/mol, respectively. Furthermore, the highest docking scores of the complex were additionally put through molecular dynamics (MD) simulation analysis. MD simulations of the selected complex were performed at 100 ns to study the stability of the genistin-ubiquitin-like protein CA 15.3 complex, which appeared to be quite stable. Additionally, the ADMET study demonstrated that genistin complies with all drug-likeness standards, including Lipinski, Egan, Veber, Ghose, and Muegge. Therefore, based on the results, genistin can be considered as one of the potential drugs for the management and treatment of BC. In addition, the obtained results suggest that genistin could pave the way for new drug discovery to manage breast cancer and has potential in the development of nutraceuticals.

Isolation and Preparation Three Types of Antigens From S.pseudintermedius

This study aimed to prepare and evaluate the immunogenic activity of three distinct antigens derived from S. pseudintermedius, a prominent isolate identified from otitis externa samples of dogs and human infections. The isolation and identification of Staphylococcus species were conducted using rigorous cultural, morphological, and biochemical methods, with confirmation via VITEK technique. A total of 100 samples, comprising 50 from humans and 50 from dogs, were collected. Following successful antigen preparation, twenty rabbits were immunized and divided into four groups, including a control group. Cellular immunity was assessed through a delayed hypersensitivity (DTH) skin test, revealing positive skin reactions among the immunized groups. Subsequent challenge dosing resulted in clinical observation, with marked signs of illness and severe infection in the control group. Histopathological examination further confirmed this infection and bacterial isolation. Remarkably, the prepared antigens exhibited substantial protein concentrations and induced a robust immune response in immunized rabbits against experimental L. monocytogenes infection. These findings underscore the potential of S. pseudintermedius antigens for developing protective strategies against bacterial infections in both veterinary and human medicine, offering significant implications for global health practitioners and researchers in the field.
 Highlights :
 
 Rigorous identification: S. pseudintermedius derived antigens evaluated after precise Staphylococcus species identification using cultural, morphological, and biochemical methods, confirmed via VITEK technique.
 Robust immune response: Immunization with prepared antigens induced positive delayed hypersensitivity skin reactions and protected against severe L. monocytogenes infection, demonstrating potential for cross-species bacterial infection prevention.
 Clinical and histopathological insights: Control group exhibited illness and Listeriosis upon challenge, validated through histopathological examination, highlighting antigen-induced protection against bacterial invasion.

DOES TRYPANOSOMA EVANSI HAVE THE MAXICIRCLE GENE, OR CAN TRYPANOSOMA EQUIPERDUM BE ISOLATED FROM BOVINES?

Identifying a trypanosome isolate is generally based on morphological observations and molecular identification of one of the genes, usually internal transcribed spacer 1 and 2 of ribosomal DNA (ITS1 rDNA, ITS2 rDNA), a variant surface glycoprotein of Rode Trypanozoon antigen type 1.2 (VSG RoTat 1.2), or expression site-associated genes (ESAG). However, this identification is insufficient because these genes cannot distinguish organisms in the subgenus Trypanozoon to the species level. A molecular approach using at least 5 sets of primers is needed, namely, ITS1, ESAG6/7, MINI, RoTat 1.2, and ND5, for stratified selection to obtain more targeted and conclusive results. Using this method to analyze isolates from Indonesia provided unexpected results: 9 isolates previously identified as Trypanozoon were found to have the kDNA maxicircle gene. Nine isolates of Trypanosoma equiperdum were identified for the first time in Indonesia, isolated from bovine (cattle and buffaloes). The identification of T. equiperdum in the 9 isolates was confirmed by analysis of the nucleotide sequence identity of the nad5-kDNA maxicircle gene.

A subunit vaccine encoding glycoprotein holds potential to protect snakehead (Channa argus) from snakehead vesiculovirus infection

Snakehead vesiculovirus (SHVV) is one of the most harmful viral diseases to the snakehead farming industry. Subunit vaccines with highly protective effects strongly depend on the type of vaccine antigen. In order to screen the potential antigen proteins with efficient immunogenicity and reactogenicity, the glycoprotein (G), matrix protein (M), nucleoprotein (N) and phosphoprotein (P) of SHVV were molecular cloned, sequences analyzed and prokaryotic expressed in this study, then four recombinant proteins were served as subunit vaccines to immunize snakehead by intraperitoneal injection to evaluate the immune effects based on the indexes including the serum antibody level, nonspecific enzyme activity, immune-related gene expression and relative percent survivals (RPS). The results showed that compared with control group, the serum antibody levels and T-AOC activities were significantly higher in all four subunit vaccine groups, the serum ACP and AKP activities in four subunit vaccine groups were higher with different degree, and the detected immune-related genes expression levels were remarkably higher in four subunit vaccine groups except IL-8 gene in spleen of rP group. Among four subunit vaccines, rG protein displayed better immune effect than other three proteins. Besides, the RPS of rG group was highest with 83.3%, followed by rM (66.7%), rN (50.0%) and rP (8.3%), respectively. The above results indicated that glycoprotein was a better candidate protective antigen of SHVV and held potential to protect Snakehead from SHVV infection in the type of subunit vaccine.

Canine Parvoviral Enteritis Incidence in Rostov-on-Don in 2017-2022: A Retrospective Analysis

Introduction. The canine parvoviral enteritis, first detected in 1978 [1], remains a widespread cause of morbidity and mortality in young dogs. The virus high virulence and resistance are attributed to its ability to mutate and undergo changes, that partly explains the continuing epidemic of the canine parvoviral enteritis.The nature of the disease itself got modified since the first time of virus detection. Although the initial outbreaks had the panzootic character implying high morbidity and mortality, nowadays the majority of adult dogs have either vaccinal immunity or immunity acquired through natural infection, thus the parvoviral infection is primarily manifested in young dogs aged from weaning to six months old as the enteric disease form.The objective of the study was to investigate the antigenic essence of the canine parvoviral enteritis by analysing the strain phylogenetic relationships in the absence of grouping the same antigenic type viruses upon emerging the mutations.Materials and Methods. The material for the study was selected by the method of continuous sampling of the medical records (by generating random numbers in the range of 1–255) and conducting interviews with the personnel (veterinary doctors) at the «Animal Clinic» veterinary hospital in Rostov-on-Don from September 2017 to October 2022. Data on the dog owners was concealed, hypotheses were not communicated to the clinic management, that excluded possible errors and conflict of interest. The study focused on the analysis of the epidemiological situation in Rostov-on-Don with regard to the canine parvoviral enteritis incidence and mortality rate, the analysis was based on the practices of the «Animal Clinic» veterinary hospital, volunteer organisations, homeless animal care foundations and «Dog Patrol» nonprofit organisation and was interpolated to the epidemiological situation in the city as a whole.The study of the canine parvoviral enteritis cases, the disease epidemiology and its combat methods, treatment, prophylaxis and the cutting-edge developments in the field was carried out based on the scientific data of the last 5 years. The objects of the study were dogs of various breeds and ages admitted to the clinic.Results and Discussion. The established phylogenetic interrelation of the divergent CPV-2b strain with the alike genetic strain-ancestor CPV-2c, indicates a complete replacement of the circulating strains, which has practical value for the veterinary science as it does not merely enable formation of a disease model for a specific region but also allows evaluating the speed and features of spreading the disease and predicting its course and mortality.Conclusion. For the southern region the parvoviral enteritis is a severe and life-threatening disease due to the rates of its overall incidence and mortality. The results of the statistical analysis of 307 medical records of the dogs diagnosed with the parvoviral enteritis in a number of Rostov-on-Don veterinary clinics revealed twice as high mortality rate in male dogs compared to females, although the distribution of the number of disease cases was relatively equal, except for the range of 30-90 days when a statistically significant difference in mortality was not registered. A separate local surge in mortality rate by over 36 %, which is not correlated either with the reduced number of cases during this period or the average level of fatal outcomes, indicates the need to correlate the patients’ immune status with the applied treatment protocols and the CPV-2c strain penetration, having the higher virulence in Europe (in 2015–2017) [2] and the European part of Russia.When analysing the «age-number of cases» data, the correlation was made with the surge in patients at the age of about 1 year old. The vaccination reduced the incidence rate in dogs and disease severity in those infected, but at the same time increased the mutation numbers in the virus genome (especially in the genes encoding the protein responsible for binding to the receptor cells [3]), which in turn led to increased virulence.

Evolution of Vaccines Formulation to Tackle the Challenge of Anti-Microbial Resistant Pathogens.

The increasing diffusion of antimicrobial resistance (AMR) across more and more bacterial species emphasizes the urgency of identifying innovative treatment strategies to counter its diffusion. Pathogen infection prevention is among the most effective strategies to prevent the spread of both disease and AMR. Since their discovery, vaccines have been the strongest prophylactic weapon against infectious diseases, with a multitude of different antigen types and formulative strategies developed over more than a century to protect populations from different pathogens. In this review, we review the main characteristics of vaccine formulations in use and under development against AMR pathogens, focusing on the importance of administering multiple antigens where possible, and the challenges associated with their development and production. The most relevant antigen classes and adjuvant systems are described, highlighting their mechanisms of action and presenting examples of their use in clinical trials against AMR. We also present an overview of the analytical and formulative strategies for multivalent vaccines, in which we discuss the complexities associated with mixing multiple components in a single formulation. This review emphasizes the importance of combining existing knowledge with advanced technologies within a Quality by Design development framework to efficiently develop vaccines against AMR pathogens.

Pathological classification of malignant peritoneal mesothelioma and comparative analysis with peritoneal carcinomatosis

Objective: To analyze the pathological classification of malignant peritoneal mesothelioma (MPeM) and screen the immunohistochemical markers that can distinguish MPeM from peritoneal metastatic carcinoma (PC) . Methods: In June 2020, the pathological results of peritoneal biopsy of 158 MPeM and 138 PC patients from Cangzhou Central Hospital, Cangzhou People's Hospital, and Cangzhou Hospital of Integrated Traditional Chinese and Western Medicine from May 2011 to July 2019 were retrospectively analyzed, and the pathological classifications of MPeM in Cangzhou were summarized. Immunohistochemical markers of MPeM and PC patients were analyzed, and receiver operating characteristic curve (ROC curve) was drawn for differential diagnosis of MPeM and PC. Results: There were 55 male and 103 female MPeM patients in Cangzhou, with an average age of 57.1 years old. The asbestos exposure rate was 91.14% (144/158). The most common pathological classifications were cutaneous type, accounting for 90.51% (143/158). There were significant differences in the expression of calreticulum protein, CK5/6, vimentin, D2-40, carcinoembryonic antigen (CEA) and tail type homologous nuclear gene transcription factor 2 (CDX-2) between MPeM and PC (P<0.05). Among the 6 positive markers, the sensitivity of calreticulum protein was the highest (0.905) and CEA was the lowest (0.428) . Conclusion: Calreticulum protein, CK5/6, vimentin, D2-40, CEA and CDX-2 may be used as specific markers to distinguish the diagnosis of MPeM from PC.

Evaluating staging laparoscopy indications for pancreatic cancer based on resectability classification and treatment strategies for patients with positive peritoneal washing cytology.

The prognosis of pancreatic ductal adenocarcinoma (PDAC) in patients with positive peritoneal washing cytology (CY1) is poor. We aimed to evaluate the results of staging laparoscopy (SL) and treatment efficacy in CY1 patients based on a resectability classification. We retrospectively reviewed 250 patients with PDAC who underwent SL before the initial treatment between 2017 and 2023 at the University of Toyama. The breakdown of cases by resectability classification was resectable (R):borderline resectable (BR):unresectable locally advanced (UR-LA) = 131:48:71 cases. The frequency of CY1 increased in proportion to the degree of local progression (R:BR:UR-LA = 20:23:34%), but the frequencies of liver metastasis or peritoneal dissemination were comparable (R:BR:UR-LA = 6.9:6.3:8.5%). Most CY1 patients received gemcitabine along with nab-paclitaxel therapy. The CY-negative conversion rates (R:BR:UR-LA = 70:64:52%) and conversion surgery rates (R:BR:UR-LA = 40:27:9%) were inversely proportional to the degree of local progression.Comparing H0P0CY1 factors for each classification, patients with H0P0CY1 had significantly more pancreatic body or tail carcinoma and tumor size ≥32 mm in R patients, whereas in BR patients, duke pancreatic monoclonal antigen type 2 (DUPAN-2) ≥ 230 U/mL was a significant factor. In contrast, no significant factors were observed in UR-LA patients. The CY1 rates, CY-negative conversion rates, and conversion surgery rates varied according to local progression. In the case of R and BR, SL could be considered in patients with pancreatic body or tail carcinoma, large tumor size, or high DUPAN-2 level. In UR-LA, SL might be considered for all patients.

Clinical significance of possible HLA biomarkers in axial spondyloarthritis beyond HLA-B27 positivity.

The aim of this study is to compare four forms of axial spondyloarthritis (axSpA): non-radiographic axial spondyloarthritis (nr-axSpA), ankylosing spondylitis (AS), non-radiographic axial psoriatic arthritis (nr-axPsA) and radiographic axial psoriatic arthritis (r-axPsA). In a cross-sectional retrospective study, gender difference, human leukocyte antigen (HLA) typing, laboratory C-reactive protein (CRP) and erythrocyte sedimentation (SE) values, and radiographic and magnetic resonance scans were analyzed. One hundred and thirty-seven patients were included in the study: 45 AS, 51 nr-axSpA, 32 r-axPsA and 9 nr-axPsA; 74 women and 63 men. Most of the gender, laboratory and radiological findings confirmed the results of previously conducted studies about each group of the investigated axSpA. The key findings of our study are the newly detected findings of HLA typing beyond HLA-27 positivity: HLA-DR16 in AS, HLA-DR11 in nr-axSpA, HLA-B13, HLA-B57, HLA-Cw12 and HLA-DR7 in r-axPsA, and HLA-B18 in nr-axPsA. Our study also confirmed some of the results of previously conducted studies on predominant genes of HLA typing in axSpA: HLA-B27 in AS, HLA-B39 and HLA-Cw6 in r-axPsA, and HLA-Cw7 in nr-axPsA. Important conclusions about the nr-axPsA group cannot be drawn because of the very small number of subjects included in this group of axSpA. Our results suggest that the newly detected HLA typing findings beyond HLA-B27 positivity could be possible biomarkers of early detection of axSpA, but further studies on larger samples are needed.

Molecular and genetic diversity in isolates of Trypanosoma evansi from naturally infected horse and dogs by using RoTat 1.2 VSG gene in Madhya Pradesh, India.

Trypanosoma evansi is a protozoan parasite that can infect a wide range of animals and is widespread around the world. In this study, we analyzed four fatal cases of T. evansi infection using clinical, parasitological, and molecular approaches. We also explored the genetic diversity, demographic history, and population-genetic structure of T. evansi using available Rode Trypanozoon antigenic type (RoTat) 1.2 gene sequences. Clinical findings of infected animals revealed high fever, anemia, weakness, and anorexia. The animals were treated with diminazene aceturate, which was moderately effective, and hematobiochemical parameters showed changes in hemoglobin and glucose levels. The molecular and genetic diversity of T. evansi was analyzed using the RoTat 1.2 VSG gene. Phylogenetic and haplotype analysis revealed two distinct clusters of T. evansi circulating in India. The genetic diversity indices, neutrality tests, gene flow, and genetic differentiation outcomes confirmed the genetic diversity of the T. evansi population, with a lack of uniformity. The identification of two distinct clusters, exhibiting differential demographic histories and evolutionary forces, implies that the clusters may have undergone independent evolutionary trajectories or experienced different environmental pressures. The present findings underlined the need of an early and precise diagnosis in order to treat and control T. evansi infections, and the RoTat 1.2 VSG gene is an important genetic marker for understanding the genetic diversity and evolutionary history of T. evansi. This knowledge can be used to create tailored strategies to control and manage the infection in an endemic region.

The prevalence of IgG antibodies against milk and milk antigens in patients with multiple sclerosis.

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS). The pathophysiology of MS is complex and is said to be influenced by multiple environmental determinants, including diet. We and others have previously demonstrated how consumption of bovine milk can aggravate disease severity in MS patients, which can be explained by molecular mimicry between milk antigens and those expressed within the CNS. In this study we set out to identify alternatives to drinking cow milk which might be less detrimental to MS patients who have a genetic predisposition towards developing antibody titers against bovine milk antigens that cross-react with CNS antigens. To this end, we screened 35 patients with MS and 20 healthy controls for their IgG reactivity against an array of animal-sourced milk, plant-based alternatives as well as individual antigens from bovine milk. We demonstrate that MS patients have a significantly higher IgG response to animal-sourced milk, especially cow milk, in comparison to healthy donors. We also show that the reactivity to cow milk in MS patients can be attributed to reactivity against different bovine milk antigens. Finally, our correlation data indicate the co-existence of antibodies to individual bovine milk antigens and their corresponding cross-reactive CNS antigens. Taken together, we suggest screening of blood from MS patients for antibodies against different types of milk and milk antigens in order to establish a personalized diet regimen.

O02 Neoantigens from actinic keratosis are predicted to be more immunogenic than those from cutaneous squamous cell carcinoma – a strategy for immune escape?

Abstract Actinic keratosis (AK) is a precursor of cutaneous squamous cell carcinoma (cSCC). More than 70% of cSCC arise from AKs, but fewer than 0.6% of AKs progress to cSCC. We have shown that the molecular landscapes of AK and cSCC are similar in terms of tumour mutational burden (TMB), copy number burden, mutational signatures and driver gene mutations but differ in levels of intrasample heterogeneity. We have also demonstrated that alterations in signalling pathways, including transforming growth factor-β, may be important for AK-to-SCC progression. However, the pathomechanisms underlying progression are not well characterized and it remains unclear why the majority of AKs do not progress, and most will regress. There is some evidence that differences in the neoantigen landscapes of AK and cSCC may play a role, and a previous study examining neoantigens in AK and cSCC from nine patients was the first to suggest that there is downregulation of the expression of highly immunogenic neoantigens in the immune escape of cSCC. In this study we further explore, in a larger data set, the hypothesis that the neoantigen recognition potential of AK vs. cSCC is a key determinant of AK-to-cSCC progression. We generated exome and transcriptome data from AK (n = 5) and cSCC (n = 15) with matched normal exome data (from blood). Human leucocyte antigen typing was performed using the OptiType algorithm and neoantigens restricted to major histocompatibility complex (MHC) class I were predicted using the NeoPredPipe workflow. Fitness of neoantigens, defined as the overall effectiveness of a neoantigen at stimulating an immune response, was determined by combining measures of MHC binding affinity and T-cell receptor recognition potential for the predicted neoantigen, and an overall neoantigen recognition potential score was derived for each neoantigen. The difference in median total neoantigen burden for AK and cSCC was not statistically significant [11 224 (range 30–17 330) and 1680 (range 3159–67 357), respectively (P = 0.07)]. Neoantigen burden was strongly correlated with TMB in both AK and cSCC. We found that, compared with cSCC, AK had predicted neoantigens with significantly higher mean MHC-binding affinity (P = 0.049), mean T-cell receptor recognition potential (P = 0.0093) and overall neoantigen recognition potential (P = 0.02). Taken together, our results confirm that neoantigens in AKs are predicted to be more effective at eliciting an immune response than those in cSCC. This suggests there may be a selection pressure in AKs for mutations that result in fewer immunogenic neoantigens resulting in immune escape and progression to cSCC. Finally, our results underscore the critical requirement for an intact immune system in prevention of AK-to-cSCC progression.

The DKMS-BMST Thalassemia program: Description, scope and impact

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only established curative therapy for thalassemia. However, access to transplantation is constrained in limited-resource settings. Human leukocyte antigen (HLA) typing of patients and potential donors is one relevant barrier to allo-HSCT. Here we report on the DKMS-BMST thalassemia program. The program runs thalassemia camps where buccal swab samples are obtained from patients and potential family donors and HLA-typed free of charge. Since 2018, within the scope of the program, 390 camps have been conducted in India and 10,216 samples have been collected and HLA-typed. We are aware of 220 transplantations in patients of the program and are confident that the program makes a small but significant contribution to the cure of patients with thalassemia in India.

Rhesus isoimmunization: An underappreciated reproductive risk

In sub-Saharan Africa, pregnancy-related complications are a significant contributor to morbidity, mortality, and rising healthcare costs (1). The membrane of human red blood cells (RBCs) may contain the Rhesus (Rh) antigen. There are several types of Rh antigens but most important is the RhD antigen. Along the whole RhD protein, a group of conformation-dependent epitopes make up the D antigen. The most clinically significant blood group antigens that determine whether fetomaternal blood compatibility exists are the RhD and ABO systems (2). While the D-negative phenotype is linked to a deletion of RHD in most Caucasians, it is uncertain why the D antigen is not expressed other populations like the Japanese and Africans where it is linked to a grossly normal RHD (3). Accordingly, if a mother is Rh D-negative and the fetus is Rh D positive, she may produce antibodies through a process called RhD sensitization if she is exposed to fetal antigens. Sensitization is unlikely to damage the index fetus in normal conditions, but it may result in recurrent pregnancy losses, an infant with sensitized RBCs or to severe types of hemolytic disease of the newborn presenting with jaundice, anemia, developmental problems, or stillbirth (4).&#x0D; Rhesus isoimmunization can be prevented, but most Sub-Saharan African countries have not fully utilized these opportunities. As a result, many women are at risk due to poor medical records following potentially sensitizing events and a lack of access to crucial diagnostic and therapeutic commodities. These have significantly increased the difficulty of managing Rh-negative pregnancies (5).&#x0D; There has been a contentious suggestion for the universal prophylactic use of anti-D immunoglobulin in all pregnancies, including those following termination or ectopic pregnancy (6). We know that only a few at-risk women will profit from this strategy, which will unquestionably be highly expensive. However, there is no question that the Rh-negative pregnant population of Africa needs to have access to anti-D immunoglobulin on a global basis (7)(8). It follows that appropriate strategies should be instituted to effectively identify at-risk women and treat them accordingly (9).&#x0D; Leaders in obstetrics practice from various African countries met towards the end of last year to discuss the unmet need to address the burden of Rhesus disease. They noted that, despite being entirely preventable, Rhesus disease was identified as one of the roadblocks to achieving maternal universal health coverage. The team recommended all stakeholders to facilitate access to anti-D immunoglobulin and screening tools for widespread screening and treatment.

Generation of the NeoThy mouse model for human immune system studies.

Humanized mouse models, created via transplantation of human hematopoietic tissues into immune-deficient mice, support a number of research applications, including transplantation immunology, virology and oncology studies. As an alternative to the bone marrow, liver, thymus humanized mouse, which uses fetal tissues for generating a chimeric human immune system, the NeoThy humanized mouse uses nonfetal tissue sources. Specifically, the NeoThy model incorporates hematopoietic stem and progenitor cells from umbilical cord blood (UCB) as well as thymus tissue that is typically discarded as medical waste during neonatal cardiac surgeries. Compared with fetal thymus tissue, the abundant quantity of neonatal thymus tissue offers the opportunity to prepare over 1,000 NeoThy mice from an individual thymus donor. Here we describe a protocol for processing of the neonatal tissues (thymus and UCB) and hematopoietic stem and progenitor cell separation, human leukocyte antigen typing and matching of allogenic thymus and UCB tissues, creation of NeoThy mice, assessment of human immune cell reconstitution and all experimental steps from planning and design to data analysis. This entire protocol takes a total of ~19 h to complete, with steps broken up into multiple sessions of 4 h or less that can be paused and completed over multiple days. The protocol can be completed, after practice, by individuals with intermediate laboratory and animal handling skills, enabling researchers to make effective use of this promising in vivo model of human immune function.

Sensitivity of Carcinoembryonic Antigen Level in Head and Neck, Breast, Lung, Genitourinary, Gastrointestinal Carcinoma

Objective: To determine the Carcinoembryonic Antigen Levels among patients with epithelial carcinoma presenting at a tertiary care hospital.&#x0D; Study Design: Prospective longitudinal study.&#x0D; Place and Duration of Study: Department of Medical Oncology, Jinnah Postgraduate Medical Centre, Karachi Pakistan, from May 2019 to May 2020.&#x0D; Methodology: Two hundred six patients presenting with a confirmed diagnosis of epithelial carcinoma, aged more than 15 years and of either gender were enrolled in the study. The CEA levels in all the patients were measured using an ELISA kit. The CEA levels were classified as 0-3.0 ng/mL, 3.1-5.0 ng/mL, 5.1-10 ng/mL and &gt;10ng/mL.&#x0D; Results: The mean age of the cancer patients was estimated as 47.30±14.10 years. Most patients had rectum carcinoma 28(13.59%), followed by CA sigmoid 25(12.14%), respectively. The median CEA level was estimated as 5.80ng/mL ranging from 0.82 to 1000 ng/mL. About 76 patients had CEA level &gt;10ng/mL (36.9%), 55 had CEA level 0-3.0 ng/mL (26.7%), 43 had CEA level 3.1-5.0ng/mL (20.9%), and 32 had CEA level 5.1-10.0ng/mL (15.5%). A statistically significant difference in proportions of CEA levels was found for histological type (p=0.001), type of cancer (p=0.001) and number of Mets (p=0.003).&#x0D; Conclusion: Rectal cancer profoundly expressed carcinoembryonic antigen levels, followed by sigmoid, colon, and ovarian cancer. Moreover, there is a significant association among carcinoembryonic antigen levels, histology, type and metastasis of cancer.

ASO Author Reflections: Prognostic Role of Preoperative Duke Pancreatic Monoclonal Antigen Type 2 Values in Patients with Pancreatic Cancer: Focusing on the Usefulness in Patients with Normal CA19-9.

Pancreatic cancer (PC) is one of the most aggressive cancer types, and carbohydrate antigen (CA) 19-9 has been the most useful biomarker for its surveillance and prognosis prediction. However, CA19-9 may not be sufficiently prognostic in some patients, such as Lewis antigen-negative phenotype (Le[a-b-]) patients who secrete little or no CA19-9. Duke pancreatic monoclonal antigen type 2 (DUPAN-2) has been proposed as a complementary marker to CA19-9 in PC patients, but its utility in Le(a-b-) patients has only been reported in a limited number of cases. In a retrospective analysis of 224 PC patients who underwent surgery, the present study investigated the utility of DUPAN-2 in combination with CA19-9. The study subjects were divided into three groups based on their CA19-9 and DUPAN-2 levels. The normal CA19-9/high DUPAN-2 group had significantly larger tumors and a higher frequency of microscopic vascular invasion, perineural invasion, and recurrence than the normal CA19-9/normal DUPAN-2 group. Both the disease-free survival and disease-specific survival (DSS) of patients in the normal CA19-9/high DUPAN-2 group were significantly shorter than those in the normal CA19-9/normal DUPAN-2 group, and comparable with those in the high CA19-9 group. The results suggest that DUPAN-2 may be useful as a complementary biomarker to CA19-9 in PC, especially in patients who have normal CA19-9 levels. However, since this was a single-center, retrospective study, multicenter studies are needed to confirm the findings and determine the optimal cut-off value for patients with normal CA19-9 levels.

Ultrarapid and high-resolution HLA class I typing using transposase-based nanopore sequencing applied in pharmacogenetic testing.

Nanopore sequencing has been examined as a method for rapid and high-resolution human leukocyte antigen (HLA) typing in recent years. We aimed to apply ultrarapid nanopore-based HLA typing for HLA class I alleles associated with drug hypersensitivity, including HLA-A*31:01, HLA-B*15:02, and HLA-C*08:01. Most studies have used the Oxford Nanopore Ligation Sequencing kit for HLA typing, which requires several enzymatic reactions and remains relatively expensive, even when the samples are multiplexed. Here, we used the Oxford Nanopore Rapid Barcoding kit, which is transposase-based, with library preparation taking less than 1h of hands-on time and requiring minimal reagents. Twenty DNA samples were genotyped for HLA-A, -B, and -C; 11 samples were from individuals of different ethnicity and nine were from Thai individuals. Two primer sets, a commercial set and a published set, were used to amplify the HLA-A, -B, and -C genes. HLA-typing tools that used different algorithms were applied and compared. We found that without using several third-party reagents, the transposase-based method reduced the hands-on time from approximately 9h to 4h, making this a viable approach for obtaining same-day results from 2 to 24 samples. However, an imbalance in the PCR amplification of different haplotypes could affect the accuracy of typing results. This work demonstrates the ability of transposase-based sequencing to report 3-field HLA alleles and its potential for race- and population-independent testing at considerably decreased time and cost.

Synthesis of structure-defined β-1,4-GlcNAc-modified wall teichoic acids as potential vaccine against methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a high priority pathogen due to its life-threating infections to human health. Development of prophylactic or therapeutic anti-MRSA vaccine is a potential approach to treat S. aureus infections and overcome the resistance crisis. β-1,4-GlcNAc glycosylated wall teichoic acids (WTAs) derived from S. aureus are a new type of antigen that is closely associated with β-lactam resistance. In this study, structure-defined β-1,4-GlcNAc-modified WTAs varied in chain length and numbers of GlcNAc modification were synthesized by an ionic liquid-supported oligosaccharide synthesis (ILSOS) strategy in high efficiency and chromatography-free approach. Then the obtained WTAs were conjugated with tetanus toxin (TT) as vaccine candidates and were further evaluated in a mouse model to determine the structure-immunogenicity relationship. In vivo immunological studies revealed that the WTAs-TT conjugates provoked robust T cell-dependent responses and elicited high levels of specific anti-WTAs IgG antibodies production associated with the WTAs structure including chain length as well as the β-1,4-GlcNAc modification pattern. Heptamer WTAs conjugate T6, carrying three copy of β-1,4-GlcNAc modified RboP, was identified to elicit the highest titers of specific antibody production. The T6 antisera exhibited the highest recognition and binding affinity and the most potent OP-killing activities to MSSA and MRSA cells. This study demonstrated that β-1,4-GlcNAc glycosylated WTAs are promising antigens for further development against MRSA.