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OBJECTIVE: To determine the Frequency of Germinal Center B (GCB) type Diffuse Large B Cell Lymphoma (DLBCL) in Karachi, Pakistan, and to screen the DLBCL patients for EZH2 mutation. METHODOLOGY: A cross-sectional study was conducted on DLBCL patients, and IHC analysis, PCR, and sequencing of the EZH2 gene were used. This study was conducted from August 2016 to July 2020. Surgical specimens received in the histopathology section of DDRRL centrally and from different collection units throughout Sindh having been diagnosed with DLBCL were collected. Three markers (CD10, BCL6, and MUM1) were used for subtyping. DNA was manually extracted from the FFPE Blocks of DLBCL cases using phenol: chloroform: isopropanol alcohol. Polymerase chain reaction (PCR) was performed, and samples were sent from Dow University to Korea (Macrogen) for sequencing. The amplified product was 189bp. Primers were used for EZH2 to flank the region containing the tyrosine domain on exon 16 and exon 18. RESULTS: Out of 116 cases, 60.3% and 39.7% were male and female, respectively. The mean age was 49.73±16.38 years. The mean survival duration of cases diagnosed with GCB was 8.21±7.39 months. Polymorphism of EZH2 mutation (Tyr 701 Asn) was found to be positive in GCB-DLBCL for three samples (16.6%). CONCLUSION: In the current study, Sanger sequencing was used to detect any mutation in the coding region of EZH2, and a unique mutation was reported (Tyr701Asn).

Background: Prediabetes is a metabolic condition characterized by blood sugar levels higher than normal but below the diabetic range. The onset of microvascular complications may set in this phase, hence intervening during the prediabetic stage becomes crucial in halting the progression to irreversible diabetes. We compared blood sugar levels between lifestyle modification and metformin medication and identified which option is better for managing prediabetes. Methods: A quasi-experimental study was done from January to October 2023 recruiting 150 prediabetic adults coming to Sikander Abad clinic through convenience sampling including 18 to 65 years of age while obese adults, those who had hypoglycemia within the past 3 months, and GDM women were excluded. Patients were divided into two groups and given a choice to choose pharmacotherapy (Metformin) or non-pharmacological measures (lifestyle modification). A thorough history and examination were done on all patients and Fasting Blood Sugar was done initially and after 3 months. SPSS version 21 was used. Independent and paired sample T-tests were used and chi-square was computed to see associations with the demographic factors. P-value

OBJECTIVE: This study evaluates the frequency and relationship between Thrombocytopenia and CRP levels in Covid-19 patients. METHODOLOGY: The cross-sectional retrospective study was conducted at Dr. Ishrat ul Ebad Khan Institute of Blood Diseases from October 2021 to September 2022. Data was collected from existing data from the blood bank and hematology laboratory. C reactive protein (CRP) and complete blood count (CBC) levels were observed in Covid-19 patients. Both genders, all age groups, COVID-19 patients and patients having Thrombocytopenia and raised levels of CRP were included in this study. At the same time, other bone marrow transplant patients and other infectious diseases such as Hepatitis B, Hepatitis C, Dengue, and Malaria were excluded from this study. Statistical analysis was done by using the SPSS 20.0 version. RESULTS: Out of 844 patients, the mean age was 50 years, and the male proportion was high at 70% (n=590). Most patients, 95% (n=803) had grade 1 thrombocytopenia. The mean age was found to be higher (>50 years) in Grades 1 and 2 as compared to Grades 3 and 4, where we found (

Background: Injury is a significant issue of public health, a major global burden and first aid training programs incorporated as part of school curriculum will have a considerable impact on public health, and this will in turn reduce out of hospital cardiac arrests. The study aimed to determine the immediate First aid knowledge and long term retention of its knowledge (after three months) of the students (13-18 years), participating in a volunteer program in Karachi. Methods: This study was conducted in a tertiary care hospital, delivering First aid training to 143 participants by Basic life support (BLS) trained staff in a tertiary care hospital. The design consisted of a pre-test, post-test and follows up assessments, carried out via questionnaire and hands-on assessment. SPSS was used for statistical analysis. ANOVA and Friedman test was applied to evaluate the dependent variable’s improvement in knowledge between pre, post and follow up tests. Results: From 143 participants, 41.5 %, 63.8 % and 61 % satisfactory results were seen in the pre-test, post-test and follow up assessments, respectively. In Pre-test, males scored 18.75 % and the females scored 8.42%, whereas, in the post-test, the scores were 75.7% and 64.58% respectively. Similarly, in follow up evaluations results obtained were 62.1% and 45.8% respectively. Hands-on scores were 88.4% in males and 95.8 % in females. Conclusion: The level of first aid knowledge was unquestionably less satisfactory in the pre-test evaluations compared to post-test and follows up assessments. Due to its effectiveness, it should be incorporated as part of the school curriculum. Keywords: Adolescents; Knowledge; Cardiopulmonary Resuscitation.

Objective: To evaluate the relevance of thrombocytopenia to other disease states in patients reporting at a haematology laboratory. Method: The cross-sectional, retrospective study was conducted at the Dow Diagnostic Research and Reference Laboratory of the Dr Ishrat ul Ebad Khan Institute of Blood Diseases, Down University of Health Sciences, Karachi, and comprised data from the blood bank and haematology laboratory related to patients who visited during October 2021to August 2022. Data was analysed using SPSS 26. Results: Of the 1,249 patient with mean age 38.5±19.7 years (range: 0-89 years), 79(6.3%) were aged 40. Most of the patients were males 793(63.5%), and 604(48.4%) had Grade 1 thrombocytopenia. Among the females, Grade 1 thrombocytopenia was found in 246(53.9%) and Grade 2 in 140(30.7%) patients compared to 358(45.1%) and 222(28%) in males, respectively (p0.05). Conclusion: Thrombocytopenia was found to be strongly associated with gender. Key Words: Thrombocytopenia, Bleeding, Dengue, Malaria.

Background:A serious worldwide medical and public health concern occurs with the dengue virus, which is also an emerging health threat. A victim can be vulnerable and acquire dengue because of his age, genetics, nutritional status, viral strain and secondary infections. Our objective was to identify the ABO blood group system's distribution and relationship in dengue fever (DF) patients.Methodology:The current study was conducted at Dr. Ishrat ul Ebad Institute of Blood Diseases Department between September 2019 and September 2020. The research work was viewed and accepted by the ethics and research committees. Case records of patients with blood systems and their relation among dengue nonstructural protein 1(NS1)antigen /dengue immunoglobulin M (IgM) and immunoglobulin G (lgG) were included in this study.Results:Among 204 cases, 82 were blood group O positive, 59 were B positive, 38 were A positive, 16 were AB positive, 4 were O negative, and 2 were blood groupA negative. The association of the ABO blood group with dengue was statistically significant (p=0.046). Blood group O+ has the highest number of cases, 66(42.9%), than controls, 16(32%).Conclusion:This study overall consummates that blood group O is linked with a greater possibility of acquiring DF in comparison with different other blood group types. There is a significant relationship between blood group and DF observed.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Extracellular vesicles (EVs) are heterogeneous populations of nano- and micro-sized vesicles secreted by various cell types. There is mounting evidence that EVs have widespread roles in transporting proteins, lipids, and nucleic acids between cells and serve as mediators of intercellular communication. EVs secreted from stem cells could function as paracrine factors, and appear to mimic and recapitulate several features of their secreting cells. EV-mediated transport of regulatory RNAs provides a novel source of trans-regulation between cells. As such, stem cells have evolved unique forms of paracrine mechanisms for recapitulating their potencies with specialized functions by transporting non-coding RNAs (ncRNAs) via EVs. This includes the dissemination of stem cell-derived EV-ncRNAs and their regulatory effects elicited in differentiation, self-renewal, pluripotency, and the induction of reparative programs. Here, we summarize and discuss the therapeutic effects of mesenchymal stem cell-derived EV-ncRNAs in the induction of intrinsic regenerative programs elicited through regulating several mechanisms. Among them, most noticeable are the EV-mediated enrichment of ncRNAs at the injury sites contributing the regulation of matrix remodeling, epithelial mesenchymal transitions, and attraction of fibroblasts. Additionally, we emphasize EV-mediated transmission of anti-inflammatory RNAs from stem cells to injury site that potentially orchestrate the resolution of the inflammatory responses and immune alleviation to better facilitate healing processes. Collectively, this knowledge indicates a high value and potential of EV-mediated RNA-based therapeutic approaches in regenerative medicine.

The contribution of non-coding RNAs, such as microRNAs (miRNAs) in regulating physiological and pathological states has been intensively elucidated during last 15 years. The discovery of circulating miRNAs (cir-miRNAs) in variety of body fluids, is, however a recent focus of interest in understanding pathophysiological states of their originating cells/organs. Yet another stimulating debate that takes miRNAs to the next level is their presence in exosomes, and this is truly interesting area of research. Exosomes are cell-derived extracellular vesicles, and are naturally equipped biological vehicles that not only enable functional transfer of miRNAs between cells (horizontal transfer) but also foster inter-organ communication, presumably guided by organ specific receptors—decorated on their surface. However, understandings on inter-organ communication elicited by tissue specific exosomal-miRNA fingerprints remain elusive. Recently, Thomou et al., has discovered that adipose tissue contributes a large fraction of adipose specific exosomal-miRNA fingerprints in blood circulation. Experimental evidence emphasize adipose tissue as major depot of cir-miRNAs that sail through blood flow and reach to distal organs—primarily in the liver, where they regulate gene expression of host tissue and elicit metabolic control. This appears to be a genetic form of adipokines (endocrine factors secreted from adipose tissue). We review such offshore metabolic insults, and make an effort to address few important missing links between miRNAs processing and their incorporation into exosomes. We provide potential perspectives on how this knowledge could be steered towards RNA-based therapeutics for monitoring complex metabolic diseases and beyond.

The process of intercellular communication seems to have been a highly conserved evolutionary process. Higher eukaryotes use several means of intercellular communication to address both the changing physiological demands of the body and to fight against diseases. In recent years, there has been an increasing interest in understanding how cell-derived nanovesicles, known as extracellular vesicles (EVs), can function as normal paracrine mediators of intercellular communication, but can also elicit disease progression and may be used for innovative therapies. Over the last decade, a large body of evidence has accumulated to show that cells use cytoplasmic extensions comprising open-ended channels called tunneling nanotubes (TNTs) to connect cells at a long distance and facilitate the exchange of cytoplasmic material. TNTs are a different means of communication to classical gap junctions or cell fusions; since they are characterized by long distance bridging that transfers cytoplasmic organelles and intracellular vesicles between cells and represent the process of heteroplasmy. The role of EVs in cell communication is relatively well-understood, but how TNTs fit into this process is just emerging. The aim of this review is to describe the relationship between TNTs and EVs, and to discuss the synergies between these two crucial processes in the context of normal cellular cross-talk, physiological roles, modulation of immune responses, development of diseases, and their combinatory effects in tissue repair. At the present time this review appears to be the first summary of the implications of the overlapping roles of TNTs and EVs. We believe that a better appreciation of these parallel processes will improve our understanding on how these nanoscale conduits can be utilized as novel tools for targeted therapies.

BACKGROUND:Permanent joint dysfunction due to bone destruction occurs in up to 50% of patients with septic arthritis. Recently, imaging technologies such as micro computed tomography (μCT) scan have been widely used for preclinical models of autoimmune joint disorders. However, the radiological features of septic arthritis in mice are still largely unknown. METHODS:NMRI mice were intravenously or intra-articularly inoculated with S. aureus Newman or LS-1 strain. The radiological and clinical signs of septic arthritis were followed for 10 days using μCT. We assessed the correlations between joint radiological changes and clinical signs, histological changes, and serum levels of cytokines. RESULTS:On days 5-7 after intravenous infection, bone destruction verified by μCT became evident in most of the infected joints. Radiological signs of bone destruction were dependent on the bacterial dose. The site most commonly affected by septic arthritis was the distal femur in knees. The bone destruction detected by μCT was positively correlated with histological changes in both local and hematogenous septic arthritis. The serum levels of IL-6 were significantly correlated with the severity of joint destruction. CONCLUSION:μCT is a sensitive method for monitoring disease progression and determining the severity of bone destruction in a mouse model of septic arthritis. IL-6 may be used as a biomarker for bone destruction in septic arthritis.

Stem cells are proposed to continuously secrete trophic factors that potentially serve as mediators of autocrine and paracrine activities, associated with reprogramming of the tumor microenvironment, tissue regeneration, and repair. Hitherto, significant efforts have been made to understand the level of underlying paracrine activities influenced by stem cell secreted trophic factors, as little is known about these interactions. Recent findings, however, elucidate this role by reporting the effects of stem cell derived extracellular vesicles (EVs) that mimic the phenotypes of the cells from which they originate. Exchange of genetic information utilizing persistent bidirectional communication mediated by stem cell-EVs could regulate stemness, self-renewal, and differentiation in stem cells and their subpopulations. This review therefore discusses stem cell-EVs as evolving communication factors in stem cell biology, focusing on how they regulate cell fates by inducing persistent and prolonged genetic reprogramming of resident cells in a paracrine fashion. In addition, we address the role of stem cell-secreted vesicles in shaping the tumor microenvironment and immunomodulation and in their ability to stimulate endogenous repair processes during tissue damage. Collectively, these functions ensure an enormous potential for future therapies.

Extracellular vesicles (EVs) are membrane enclosed micro- and nano-sized vesicles that are secreted from almost every species, ranging from prokaryotes to eukaryotes, and from almost every cell type studied so far. EVs contain repertoire of bioactive molecules such as proteins (including enzymes and transcriptional factors), lipids, carbohydrates and nucleic acids including DNA, coding and non-coding RNAs. The secreted EVs are taken up by neighboring cells where they release their content in recipient cells, or can sail through body fluids to reach distant organs. Since EVs transport bioactive cargo between cells, they have emerged as novel mediators of extra- and intercellular activities in local microenvironment and inter-organ communications distantly. Herein, we review the activities of EV-associated matrix-remodeling enzymes such as matrix metalloproteinases, heparanases, hyaluronidases, aggrecanases, and their regulators such as extracellular matrix metalloproteinase inducers and tissue inhibitors of metalloproteinases as novel means of matrix remodeling in physiological and pathological conditions. We discuss how such EVs act as novel mediators of extracellular matrix degradation to prepare a permissive environment for various pathological conditions such as cancer, cardiovascular diseases, arthritis and metabolic diseases. Additionally, the roles of EV-mediated matrix remodeling in tissue repair and their potential applications as organ therapies have been reviewed. Collectively, this knowledge could benefit the development of new approaches for tissue engineering.

Extracellular vesicles (EVs) are nanosized vesicles secreted from virtually all cell types and are thought to transport proteins, lipids and nucleic acids including non-coding RNAs (ncRNAs) between cells. Since, ncRNAs are central to transcriptional regulation during developmental processes; eukaryotes might have evolved novel means of post-transcriptional regulation by trans-locating ncRNAs between cells. EV-mediated transportation of regulatory elements provides a novel source of trans-regulation between cells. In the last decade, studies were mainly focused on microRNAs; however, functions of long ncRNA (lncRNA) have been much less studied. Here, we review the regulatory roles of EV-linked ncRNAs, placing a particular focus on lncRNAs, how they can foster dictated patterns of trans-regulation in recipient cells. This refers to envisaging novel mechanisms of epigenetic regulation, cellular reprogramming and genomic instability elicited in recipient cells, ultimately permitting the generation of cancer initiating cell phenotypes, senescence and resistance to chemotherapies. Conversely, such trans-regulation may introduce RNA interference in recipient cancer cells causing the suppression of oncogenes and anti-apoptotic proteins; thus favoring tumor inhibition. Collectively, understanding these mechanisms could be of great value to EV-based RNA therapeutics achieved through gene manipulation within cancer cells, whereas the ncRNA content of EVs from cancer patients could serve as non-invasive source of diagnostic biomarkers and prognostic indicators in response to therapies.

Self-associating split fluorescent proteins (FPs) are split FPs whose two fragments spontaneously associate to form a functional FP. They have been widely used for labeling proteins, scaffolding protein assembly and detecting cell-cell contacts. Recently developments have expanded the palette of self-associating split FPs beyond the original split GFP1-10/11. However, these new ones have suffered from suboptimal fluorescence signal after complementation. Here, by investigating the complementation process, we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution. The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness, facilitating the tagging of endogenous proteins by gene editing. Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP) for multiplexed visualization of neuronal synapses in living C. elegans, demonstrating its broad applications.

OBJECTIVE: This study evaluates the frequency and relationship between Thrombocytopenia and CRP levels in Covid-19 patients. METHODOLOGY: The cross-sectional retrospective study was conducted at Dr. Ishrat ul Ebad Khan Institute of Blood Diseases from October 2021 to September 2022. Data was collected from existing data from the blood bank and hematology laboratory. C reactive protein (CRP) and complete blood count (CBC) levels were observed in Covid-19 patients. Both genders, all age groups, COVID-19 patients and patients having Thrombocytopenia and raised levels of CRP were included in this study. At the same time, other bone marrow transplant patients and other infectious diseases such as Hepatitis B, Hepatitis C, Dengue, and Malaria were excluded from this study. Statistical analysis was done by using the SPSS 20.0 version. RESULTS: Out of 844 patients, the mean age was 50 years, and the male proportion was high at 70% (n=590). Most patients, 95% (n=803) had grade 1 thrombocytopenia. The mean age was found to be higher (>50 years) in Grades 1 and 2 as compared to Grades 3 and 4, where we found (<40 years). A significant (p=0.002) mean age difference among grades was found. No significant (p=0.749) median difference in CRP among Grades were found. CONCLUSION: The current study shows that COVID-19 significantly increases C-reactive protein levels. While treating and managing the patients of COVID-19, it is essential to detect the biomarkers that can predict the severity and prognosis of this disease. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To assess the key performance indicators and the laboratory errors in the hematology laboratory. METHODOLOGY: A cross-sectional study was conducted at the DDRRL Hematology laboratory from October 2021 to September 2022. Data from the blood bank and hematological laboratory were collected. The primary investigator managed every step of the data-gathering operation. All the tools required for the process were gathered and arranged on a tray or trolley to be safe, accessible, and readily visible. Sterile glass or plastic tubes with rubber covers were used for blood collection. The information acquired during the study was inserted into a computer using an Excel sheet. SPSS version 20 was used for the analysis. All samples from the hematology section of DDRRL were included in this study, while all error-free samples were excluded. RESULT: From July to December, 414,400 hematology request forms were collected from OPD, private wards, IPD and Emergency. Overall, 2376 (0.573%) hematology laboratory errors were detected, of which 419 were Pre-analytical errors, 122 were analytical, and 1834 were post-analytical errors. CONCLUSION: We can lower the errors' likelihood if we appropriately handle the pre-analytical variables. Additionally, care must be taken when training workers to minimize the possibility of mistakes. [ABSTRACT FROM AUTHOR]