Protein S Deficiency with Favourable Foetomaternal Outcome: A Case Report

Protein S is a multifunctional plasma protein, whose deficiency, results in a rare congenital thrombophilia, inherited in an autosomal dominant pattern. It can aggravate the hypercoagulable state of pregnancy, when it presents in parallel with the condition, leading to adverse maternal outcomes and foetal loss. A 35-year-old female third gravida having previous 2 deliveries by Lower Segment Caesarean Section (LSCS) presented to emergency at 10 weeks pregnancy with chief complaints of pain and swelling in left thigh since 4-5 days. After thorough investigations and work-up, the patient was diagnosed with Protein S deficiency. She was managed conservatively and was delivered by elective LSCS with bilateral tubal ligation at 38 weeks of gestation with good foetal and maternal outcomes.The rarity of Protein S deficiency along with the successful outcome of the pregnancy makes this a unique case.Copyright © 2023 Journal of Clinical and Diagnostic Research. All rights reserved.

Active COVID-19 infection as a barrier to postpartum tubal ligation.

OBJECTIVES: To identify patients unable to obtain postpartum bilateral tubal ligations (ppBTLs) due to policy prohibiting "elective" procedures for COVID-positive patients at a single academic medical center in New Jersey. RESULTS: Upon retrospective chart review of patients requesting ppBTLS, of 110 ppBTL requests between identified via retrospective chart review between February 1, 2020, and February 28, 2022, 24 (22%) were canceled due to COVID infection. Of these patients, 10 (42%) were uninsured, 13 (54%) had Medicaid, and 22 (92%) were Hispanic/Latinx. Postpartum, five (21%) obtained interval tubal ligation, seven (37%) never received contraception, and one had a future pregnancy. CONCLUSIONS: This policy affected uninsured patients by preventing access to permanent contraception.

An ultra-sensitive one-pot RNA-templated DNA ligation rolling circle amplification-assisted CRISPR/Cas12a detector assay for rapid detection of SARS-CoV-2.

Rapid, sensitive, and one-pot diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an extremely important role in point-of-care testing (POCT). Herein, we report an ultra-sensitive and rapid one-pot enzyme-catalyzed rolling circle amplification-assisted CRISPR/FnCas12a assay, termed OPERATOR. OPERATOR employs a single well-designed single-strand padlock DNA, containing a protospacer adjacent motif (PAM) site and a sequence complementary to the target RNA which procedure converts and amplifies genomic RNA to DNA by RNA-templated DNA ligation and multiply-primed rolling circle amplification (MRCA). The MRCA amplicon of single-stranded DNA is cleaved by the FnCas12a/crRNA complex and detected via a fluorescence reader or lateral flow strip. OPERATOR presents outstanding advantages including ultra-sensitivity (1.625 copies per reaction), high specificity (100%), rapid reaction speed (∼30 min), easy operation, low cost, and on-spot visualization. Furthermore, we established a POCT platform by combining OPERATOR with rapid RNA release and a lateral flow strip without professional equipment. The high performance of OPERATOR in SARS-CoV-2 tests was confirmed using both reference materials and clinical samples, and the results suggest that is readily adaptable for point-of-care testing of other RNA viruses.

Present status of microfluidic PCR chip in nucleic acid detection and future perspective

Polymerase chain reaction (PCR) amplifies specific fragment of DNA molecules and has been extensively applied in fields of pathogens and gene mutation detection, food safety and clinical diagnosis which on the other hand, holds the drawbacks of large size instrument, high heat dissipation etc. It has been demonstrated that microfluidics technique coupling with PCR reaction exhibits characteristics of integration, automatization, miniaturization, and portability. Meanwhile, various designed fabrication of microchip could contribute to diverse applications. In this review, we summarized major works about a variety of microfluidic chips equipped with several kinds of PCR techniques (PCR, RT-PCR, mPCR, dPCR) and detection methods like fluorescence, electrochemistry, and electrophoresis detection. The development and application of PCR-based microfluidic chip in pathogen and gene mutation detection, diseases prevention and diagnosis, DNA hybridization and low-volume sample treatment were also discussed. Copyright © 2022 Elsevier B.V.

Aptamer-Assisted Proximity Ligation Assay for Sensitive Detection of Infectious Bronchitis Coronavirus.

Infectious bronchitis virus (IBV) is a coronavirus responsible for major health problems in the poultry industry. New virus strains continue to appear, causing large economic losses. To develop a rapid and accurate new quantitative assay for diagnosis of the virus without DNA extraction, we selected highly specific single-stranded DNA (ssDNA) aptamers with a high affinity to IBV, using the systematic evolution of ligands by exponential enrichment (SELEX) technology for aptamer screening, followed by high-throughput sequencing technology. Two of these aptamers, AptIBV5 and AptIBV2, were used to establish homogenous and solid-phase proximity ligation assays (PLAs). The developed assays were evaluated for their sensitivity and specificity using collected field samples and then compared to the newly developed sandwich enzyme-linked aptamer assay (ELAA) and reverse transcription-quantitative PCR (qRT-PCR), as the gold-standard method. The solid-phase PLA showed a lower limit of detection and a broader dynamic range than the two other assays. The developed technique may serve as an alternative assay for the diagnosis of IBV, with the potential to be extended to the detection of other important animal or human viruses. IMPORTANCE Infectious bronchitis virus (IBV) causes high morbidity and mortality and large economic losses in the poultry industry. The virus has the ability to genetically mutate into new IBV strains, causing devastating disease and outbreaks. To better monitor the emergence of this virus, the development of a rapid and highly sensitive diagnostic method should be implemented. For this, we generated aptamers with high affinity and specificity to the IBV in an ssDNA library. Using two high-affinity aptamers, we developed a sandwich ELAA and a very sensitive aptamer-based proximity ligation assay (PLA). The new assay showed high sensitivity and specificity and was used to detect IBV in farm samples. The PLA was compared to the newly developed sandwich ELAA and qRT-PCR, as the gold-standard technique.

Ligation-based assay for variant typing without sequencing: Application to SARS-CoV-2 variants of concern.

BACKGROUND: COVID-19 prevalence has remained high throughout the pandemic with intermittent surges, due largely to the emergence of genetic variants, demonstrating the need for more accessible sequencing technologies for strain typing. METHODS: A ligation-based typing assay was developed to detect known variants of severe acute respiratory syndrome virus 2 (SARS-CoV-2) by identifying the presence of characteristic single-nucleotide polymorphisms (SNPs). General principles for extending the strategy to new variants and alternate diseases with SNPs of interest are described. Of note, this strategy leverages commercially available reagents for assay preparation, as well as standard real-time polymerase chain reaction (PCR) instrumentation for assay performance. RESULTS: The assay demonstrated a combined sensitivity and specificity of 96.6% and 99.5%, respectively, for the classification of 88 clinical samples of the Alpha, Delta, and Omicron variants relative to the gold standard of viral genome sequencing. It achieved an average limit of detection of 7.4 × 104 genome copies/mL in contrived nasopharyngeal samples. The ligation-based strategy performed robustly in the presence of additional polymorphisms in the targeted regions of interest as shown by the sequence alignment of clinical samples. CONCLUSIONS: The assay demonstrates the potential for robust variant typing with performance comparable with next-generation sequencing without the need for the time delays and resources required for sequencing. The reduced resource dependency and generalizability could expand access to variant classification information for pandemic surveillance.

Impact of COVID-19 on endoscopic follow-up of gastroesophageal varices.

Background: Portal hypertension is considered as a major complication of liver cirrhosis. Endoscopy plays a main role in managing of gastrointestinal complications of portal hypertension. Endoscopists are at increased risk for COVID-19 infection because upper gastrointestinal (GI) endoscopy is a high-risk aerosol-generating procedure and may be a potential route for COVID-19. Objectives: To compare the outcome between cirrhotic patients who underwent classic regular endoscopic variceal ligation after primary bleeding episode every 2-4 weeks, and those presented during the era of COVID-19 and their follow-up were postponed 2 months later. Methods: This retrospective study included cross-matched 238 cirrhotic patients with portal hypertension presented with upper GI bleeding, 112 cirrhotic patients presented during the era of COVID19 (group A) underwent endoscopic variceal ligation, another session after 2 weeks and their subsequent follow-up was postponed 2 months later, and 126 cirrhotic patients as control (group B) underwent regular endoscopic variceal band ligation after primary bleeding episode every 2-4 weeks. Results: Eradication of varices was achieved in 32% of cases in group A, and 46% in group was not any statistically significant (p > 0.05); also, there was no any statistical significant difference between both groups regarding occurrence of rebleeding, post endoscopic symptoms, and mortality rate (p > 0.05). Conclusion: Band ligation and injection of esophageal and gastric vary every 2 months were as effective and safe as doing it every 2 to 4 weeks after primary bleeding episode for further studies and validation.

Rubber band ligation (RBL) for haemorrhoids. The experience of three surgical units

Haemorrhoids are the most common proctological disorder with a high incidence per year and a prevalence up to 39% in the general population (1). This condition often leads to disruption in an individual's personal and working life. Management has considerable cost implications, and therefore, economic consequences( 2). Due to the fear of SARS-COVID infection the most of patients actually regret hospitalization for surgery and choose to delay the time of treatment. RBL can be proposed as successful procedure to patients with II-III grade with a short stay in the hospital. Treatment consists initially of conservative measures such as lifestyle advice, diet and toilet behaviour. When conservative hemorrhoid therapy is ineffective, many physicians may choose other non-surgical modalities rubber band ligation, injection sclerotherapy, cryotherapy, manual dilation of the anus infrared photocoagulation, bipolar diathermy( 3), direct current electrocoagulation( 4). Rubber band ligation (RBL) was established as one of the most important, cost-effective and commonly used treatments for first-to third-degree internal hemorrhoids. It is a very effective non-surgical treatment for internal hemorrhoids. causing fibrosis, retraction, and fixation of the hemorrhoidal cushions. Rubber band ligation is also more effective than sclerotherapy and infra-red coagulation, but more painful. Overall complications occur in less than 10%. A retrospective study of 186 patients outpatients who underwent RBL with a minimum follow-up of 12 months is reported. Results confirmed it is effective until 1 year with a low rate of complications and could be offered as conservative treatment for I to III grade hemorrhoids.

The Distinctive Activation of Toll-Like Receptor 4 in Human Samples with Sepsis.

Clinical success of Toll-Like receptor-4 (TLR-4) antagonists in sepsis therapy has thus far been lacking. As inhibition of a receptor can only be useful if the receptor is active, stratification of patients with active TLR-4 would be desirable. Our aim was to establish an assay to quantify phosphorylated TLR-4 using the proximity ligation assay (PLA). HEK293 TLR4/MD2/CD14 as well as THP-1 cells were stimulated with LPS and the activation of TLR-4 was measured using the PLA. Furthermore, peripheral blood mononuclear cells (PBMCs) from 25 sepsis patients were used to show the feasibility of this assay in clinical material. Activation of TLR-4 in these samples was compared to the PBMCs of 11 healthy individuals. We could show a transient activation of TLR-4 in both cell lines. Five min after the LPS stimulation, the signal increased 6.7-fold in the HEK293 cells and 4.3-fold in the THP-1 cells. The assay also worked well in the PBMCs of septic patients. Phosphorylation of TLR-4 at study inclusion was 2.9 times higher in septic patients compared to healthy volunteers. To conclude, we established a diagnostic assay that is able to quantify the phosphorylation of TLR-4 in cell culture and in clinical samples of sepsis patients. This makes large-scale stratification of sepsis patients for their TLR-4 activation status possible.

Alterations in heparan sulfate proteoglycan synthesis and sulfation and the impact on vascular endothelial function.

The glycocalyx attached to the apical surface of vascular endothelial cells is a rich network of proteoglycans, glycosaminoglycans, and glycoproteins with instrumental roles in vascular homeostasis. Given their molecular complexity and ability to interact with the intra- and extracellular environment, heparan sulfate proteoglycans uniquely contribute to the glycocalyx's role in regulating endothelial permeability, mechanosignaling, and ligand recognition by cognate cell surface receptors. Much attention has recently been devoted to the enzymatic shedding of heparan sulfate proteoglycans from the endothelial glycocalyx and its impact on vascular function. However, other molecular modifications to heparan sulfate proteoglycans are possible and may have equal or complementary clinical significance. In this narrative review, we focus on putative mechanisms driving non-proteolytic changes in heparan sulfate proteoglycan expression and alterations in the sulfation of heparan sulfate side chains within the endothelial glycocalyx. We then discuss how these specific changes to the endothelial glycocalyx impact endothelial cell function and highlight therapeutic strategies to target or potentially reverse these pathologic changes.

Expedient Synthesis of Ubiquitin‐like Protein ISG15 Tools through Chemo‐Enzymatic Ligation Catalyzed by a Viral Protease Lbpro

Ubiquitin (Ub)‐like protein ISG15 (interferon‐stimulated gene 15) regulates innate immunity and links with the evasion of host response by viruses such as SARS‐CoV‐2. Dissecting ISGylation pathways recently received increasing attention which can inform related disease interventions, but such studies necessitate the preparation and development of various ISG15 protein tools. Here, we find that the leader protease (Lbpro) encoded by foot‐and‐mouth disease virus can promote ligation reactions between recombinant ISG15 and synthetic glycyl compounds, generating protein tools such as ISG15‐propargylamide and ISG15‐rhodamine110, which are needed for cellular proteomic studies of deISGylases, and the screening and evaluation of inhibitors against SARS‐CoV‐2 papain‐like protease (PLpro). Furthermore, this strategy can be also used to load ISG15 onto the lysine of a synthetic peptide through an isopeptide bond, and prepare Ub and NEDD8 (ubiquitin‐like protein Nedd8) protein tools.

Improving outcome in pregnancy after MI: A case study

Introduction: Coronary artery disease complicates 0.01% of pregnancies. In this case study, we illustrate the successful management strategy of a pregnancy with a cardiometabolic disorder who conceived after a ST-elevation myocardial infarction (STEMI). Case Description: A 41-year-old woman in her third pregnancy was referred to our maternal-fetal-medicine unit at 8 weeks of gestation. She had a history of an acute inferior myocardial infarction (MI) Killip 1 with 70% occlusion of the left anterior descending artery and ectatic vessels, which was successfully thrombolysed 1 year prior. On presentation, she had uncontrolled type 2 diabetes, chronic hypertension on treatment, and was morbidly obese with a BMI of 44 kg/m2. She had 2 previous lower segment caesarean sections and an umbilical hernia repair. A systematic effort involving a multidisciplinary team, both at tertiary and community level, was coordinated from the start, which successfully prevented any cardiac events during pregnancy. She recovered from Covid-19 category 4a at 28 weeks with no cardio-respiratory implications. She went on to have a lower segment caesarean section near term with tubal ligation. Post-operative recovery was uneventful, and she continued to see cardiologists and endocrinologists. Discussion: Specific risks such as MI recurrence, pre-eclampsia and low birth weight baby can be minimised through comprehensive antenatal and perinatal plans supported by patient compliance. Our case showed that pregnancy after MI results in good maternal-fetal outcome provided the pre-pregnancy cardiac performance status is good, early multidisciplinary management, patient-centred approach, close antenatal monitoring, adequate delivery preparation and patient compliance.

Expedient Synthesis of Ubiquitin-like Protein ISG15 Tools through Chemo-Enzymatic Ligation Catalyzed by a Viral Protease Lbpro.

Ubiquitin (Ub)-like protein ISG15 (interferon-stimulated gene 15) regulates innate immunity and links with the evasion of host response by viruses such as SARS-CoV-2. Dissecting ISGylation pathways recently received increasing attention which can inform related disease interventions, but such studies necessitate the preparation and development of various ISG15 protein tools. Here, we find that the leader protease (Lbpro ) encoded by foot-and-mouth disease virus can promote ligation reactions between recombinant ISG15 and synthetic glycyl compounds, generating protein tools such as ISG15-propargylamide and ISG15-rhodamine110, which are needed for cellular proteomic studies of deISGylases, and the screening and evaluation of inhibitors against SARS-CoV-2 papain-like protease (PLpro). Furthermore, this strategy can be also used to load ISG15 onto the lysine of a synthetic peptide through an isopeptide bond, and prepare Ub and NEDD8 (ubiquitin-like protein Nedd8) protein tools.

Comparative Study between Ligasure and Conventional Vessel Ligation in Thyroidectomy “Randomized-Controlled Study“

Background: Remarkable increase in incidence of thyroid cancer over previous decade has resulted in increased need for thyroid surgery. Aim and objectives: Goal of research was to evaluate safety and efficacy of using vessel sealing device ligasure (COVIDIEN®) in terms of intraoperative and postoperative complications, comparing to standard vascular ligation in open thyroid surgery. Patients and methods: A randomized-controlled trials that was conducted at Department of General Surgery of Assiut University Hospitals. A total of 80 patients were scheduled for thyroidectomy participated in the research. Those patients were randomly subdivided into 2 categories. Category 1 included cases who underwent conventional vessel ligation (n= 40 patient) and category 2 included cases who underwent ligasure vessel sealing (n=40 patients). The study was conducted in the period between 2019 and 2021. Results: Both groups had insignificant difference regarding overall recorded complications (8 (20%) vs. 7 (17.5%);p= 0.51). One patient in each group developed hoarseness while recurrent transient laryngeal nerve damage was recorded in two patients in each category. The most frequent complication was transient hypocalcaemia (12.5% of conventional ligation and 10% of ligasure sealing group). Conclusion: Ligature was superior to conventional approach in terms of lowering operation time, perioperative and postoperative blood loss, and parathyroid damage. RLN injuries, on the other hand, were comparable in both categories. Although shorter operative periods led to lower operating room occupancy expenses, overall cost of surgery was considerably greater in ligasure category. © 2022, Ain Shams University Faculty of Medicine. All rights reserved.

Total Chemical Synthesis of a SARS-CoV-2 Miniprotein Inhibitor LCB1.

Native chemical ligation is a widely used technique for peptide fragment condensation in aqueous solutions, which has broken through the length limitation of traditional solid-phase peptide synthesis. It can achieve high-efficient chemical synthesis of proteins containing more than 300 amino acid residues. Peptide hydrazide, as a valuable reagent equivalent to a thioester peptide, can be easily and efficiently prepared by the Fmoc-based SPPS method and has been widely used in native chemical ligation. Here we take the chemical synthesis of a SARS-CoV-2 miniprotein inhibitor LCB1 as an example to describe the detailed procedure of hydrazide-based native chemical ligation.

Arteriovenous Malformation of Upper Lip - a short cure for gross deformity

Aim: To evaluate the safety and success of facial artery ligation alone in arteriovenous malformation of upper lip in non-affording patients. Methodology: This prospective study was conducted at Department of Plastic surgery, on 11 consecutive patients with recurrent upper lip arteriovenous malformation who couldn’t afford angioembolization and frequent hospital visits for staged procedures. We did debulking of lesion after facial artery ligation and cosmetic lip correction. Results: Total 11 patients (9 males and 2 females) were included in this study. Majority of patients showed satisfactory results with facial artery ligation alone. One patient lost the follow up. No significant complication was noted in any patient. No recurrence was noted at 6 months and 1 year follow-up. Patient satisfaction rate remained 8.6±0.96 as per VAC. Conclusion: facial artery ligation alone provides an easy and approachable option for arteriovenous malformation where cost affordability for angioemboization and repeated hospital visits are main limitations.

Reducing Friction in Orthodontic Brackets: A Matter of Material or Type of Ligation Selection? In-Vitro Comparative Study.

(1) Background: Orthodontic appliances have changed and improved with the increasing demand for orthodontic treatment of the general population. Patients desire for shorter orthodontic treatments and for the wearing of more aesthetic devices has led to the technological development of orthodontic brackets; these were manufactured from aesthetic materials (ceramics, composite polymers) and presented different designs regarding the way archwires are ligated to the bracket. The aim of this study was to determine whether there were any differences between the static frictional forces generated by stainless steel (metallic) and polycrystalline alumina (ceramics) conventional and self-ligating brackets. (2) Methods: Static friction assessment was carried out in vitro with a universal testing machine, HV-500N-S (Schmidt Control Instruments, Hans Schmidt & Co. GmbH), intended for measuring compression and traction forces. (3) Results: The study revealed significant differences in static frictional forces at the bracket-archwire interface between the tested brackets. Stainless steel brackets produced lower static friction forces than polycrystalline alumina and self-ligating brackets generally produced lower static frictional forces than conventional brackets. The reduction of frictional forces was noticeable in the first stages of treatment, when thin, flexible orthodontic archwires (0.016" NiTi) are used. Engaged with large rectangular stainless steel archwires, (0.019 × 0.025" SS), the frictional forces produced by conventional and self-ligating metal brackets were similar, no significant differences being observed between the two types of metallic design. However, in the case of tested ceramic brackets, the results showed that the self-ligating type allows a reduction in frictional forces even in advanced stages of treatment compared to conventionally ligation. (4) Conclusions: From the perspective of an orthodontic system with low frictional forces, metal brackets are preferable to aesthetic ones, and self-ligating ceramic brackets are preferable to conventional ceramic brackets.

Positive Role of Delta Neutrophil Index (DNI) as a Prodiagnostic Marker in Cecal Ligation and Puncture (CLP)-Induced Sepsis Murine Model.

Sepsis is an emergent infectious disease and a leading cause of death despite immediate intervention. While Delta neutrophil index (DNI) and myeloperoxidase (MPO) are known as a prodiagnostic marker of sepsis, the preclinical evidence of the best marker of sepsis is unclear. For this, using a well-designed cecal ligation and puncture (CLP)-induced sepsis mouse model, we comparatively measured the level and cost-effectiveness of sepsis biomarkers such as DNI, myeloperoxidase (MPO), procalcitonin (PCT), and tumor necrosis factor-alpha (TNF-α). First, we found that the optimal time point for early detection is at 6 h, 24 h post-CLP. Strikingly, the peak level and fold change of DNI was revealed at 24 h, further showing the best fold change as compared with other biomarker levels. Given the fold change at 6, 24 h, PCT was next to DNI. Third, a cost-effectiveness survey showed that DNI was the best, with PCT next. Further, DNI level was moderate positively associated with PCT (ρ = 0.697, p = 0.012) and TNF-α (ρ = 0.599, p = 0.040). Collectively, these data indicate that DNI in CLP-induced sepsis mice is as effective as the existent inflammatory biomarkers such as MPO, PCT and TNF-α to predict the prognosis of sepsis. This might have clinically important implications that DNI is cost effective, thus quickly and rationally applying to diverse types of imminent sepsis regardless of species. This might be the first report on the validity of DNI in preclinical CLP-induced murine sepsis.

Retrospective study of online ligation student practice incorporating flipped learning into e-learning.

PURPOSE: Flipped learning has been applied in various fields, including medical professional education. E-learning is compatible with flipped learning; however, it is considered to be unsuitable for providing training on surgical techniques. In this study, we retrospectively examined the ligation performance of online students who underwent training with flipped learning incorporated into e-learning. METHODS: We conducted a retrospective study of the ligation practices of online students at the Department of General Surgery from March 2020 to June 2021. The subjects included 134 fourth- and fifth-year medical students from Gunma University School of Medicine. We conducted mid-term checks on the 8th day of practice and an examination on the 19th day. Two instructors independently evaluated and calculated scores using the original Global Rating Scale of Gunma University. We also conducted a questionnaire survey on the ligation practice of online students. RESULTS: The total average score of the three tasks was 12.4 for Instructor 1 and 12.0 for Instructor 2. All students had a passing score. The questionnaire survey showed that 70% of the students were trained in ligation at the time of the first evaluation. CONCLUSIONS: Our online training materials and training methods enabled the acquisition of ligation skills by students who had not previously received ligation training.