Staining Reaction Research Articles

Comparative analysis of different water samples as Buffer in Leishman stain.

Staining of blood films remains one of the most essential part of haematology. The different blood cells have different intracellular structure that take up stains according to chemical nature. Leishman stain is one of the Romanowsky stains used in the haematology laboratory for the staining of blood films. It contains methylene blue and eosin dye prepared in an alcohol medium and is diluted with a buffer. Phosphate buffer is used during Leishman stain as it acts as a mordant enhancing staining reactions and it is the recommended and best buffer for staining. Aim This comparative study was aimed at determining if different water samples used as buffer show significant effects on the morphology of blood cells during Leishman staining. Methods pH analyses of different water sources such as water from borehole, waterboard, rainwater, fridge water, distilled water and air conditioned water were carried out and used as buffers to carry out Leishmann staining on blood films. The stained slides were examined macroscopically and microscopically using 100x objective. Results The study has shown that deionized and tap water used as buffer could produce moderately good film with well stained red cells, white cells and platelets. Rainwater, distilled, fridge and air conditioned water showed a significant change in the morphology of blood cells both in an apparently healthy film and a diseased film Conclusion This study demonstrates that deionized water and tap water devoid of chlorine can be used as substitutes in the right pH of 6.8 in a situation where phosphate buffer is not available. It is therefore recommended that quality control should be carried out to check the quality of stains and buffer used for staining films in order to produce quality results.

PTEN is recognized as a prognostic-related biomarker and inhibits proliferation and invasiveness of skull base chordoma cells

ObjectiveThis work aimed to examine the function of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in skull base chordoma (SBC) at the clinical and cellular levels.MethodsTotally 65 paraffin-embedded and 86 frozen specimens from 96 patients administered surgery were analyzed. Immunohistochemical staining and quantitative real-time polymerase chain reaction were performed, and the associations of PTEN expression with clinical features were assessed. At the cellular level, PTEN was knocked down by the siRNA approach in the UCH-1 cell line, and cell proliferation and invasion were detected by the CCK-8 and migration assays, respectively.ResultsAt the protein level, PTEN expression was increased in non-bone-invasive tumor samples in comparison with bone-invasive specimens (p = 0.025), and elevated in soft SBCs in comparison with hard tumors (p = 0.017). Increased PTEN protein expression was associated with decreased risk of tumor progression (p = 0.002; hazard ratio = 0.981, 95% confidence interval: 0.969–0.993). At the gene expression level, the cut-off value was set at 10.5 after ROC curve analysis, and SBC specimens were divided into two groups: PTEN high group, ΔCt value below 10.5; PTEN low group, ΔCt value above 10.5. In multivariate regression analysis of PFS, the risk of tumor progression was increased in PTEN low group tumors in comparison with PTEN high group SBCs (p = 0.006). In the CCK-8 assay, in comparison with control cells, PTEN knockdown cells had increased absorbance, suggesting elevated cell proliferation rate. In the invasion assay, the number of tumor cells penetrating into the lower chamber was significantly increased in the PTEN knockdown group compared with control cells.ConclusionsDecreased PTEN expression in SBC, at the protein and gene levels, is associated with reduced PFS. PTEN knockdown in chordoma cells led to enhanced proliferation and invasiveness.

Acute thermal stress and endotoxin exposure modulate metabolism and immunity in marine mussels (Perna canaliculus)

Mass mortalities of New Zealand Green-lipped mussels (Perna canaliculus) are thought to be associated with increased water temperatures and immune challenges from opportunistic pathogens. However, the combined effects of acute thermal stress and immune stimulation on mussels are poorly understood. To investigate these responses, adult mussels were exposed to different temperatures (26 °C [thermal stress] vs 15 °C [ambient]) and a bacterial-derived endotoxin injection (with vs without) to mimic a pathogen infection. Various immunological and metabolic parameters were measured over two days via enzyme staining reactions, flow cytometry, and metabolomic profiling. None of the treatments impacted total and differential haemocyte counts, haemocyte viability or production of reactive oxygen species. Acid phosphatase and phenoloxidase activities were detected only within granulocytes (not in hyalinocytes), although their relative expressions also were not affected. Conversely, metabolite profiling exposed impacts of thermal stress and endotoxin exposure at a metabolic level, indicative of physiological changes in energy expenditure and partitioning. At the higher water temperature, free fatty acid and amino acid constituents increased and decreased, respectively, which supports an elevated energy demand and higher metabolic rate due to thermal stress. Ultimately, energy production is being sustained via multiple routes including the glycolysis pathway, TCA cycle, and β-oxidation. Additionally, branched-chain amino acids, the urea cycle and the glutathione pathway were affected by the higher temperature. The metabolic response of mussels exposed to endotoxin exposure resulted in increased metabolite response largely linked to protein and lipid degradation. After 5 days of exposure, survival data confirmed a severe physiological impact of the higher temperature through incidences of mortality. However, the thermal challenge in combination with the specific endotoxin treatment applied did not lead to a synergistic effect on mortality. These findings provide new insights into the relationship between thermal stress and immunity to better understand the immune defence system in mussels.

Tuberculosis in free-ranging and captive wild animals: Pathological and molecular diagnosis with histomorphological differentiation of granulomatous lesions

Tuberculosis (TB) is a serious zoonotic threat, impacting the human-livestock-wildlife interface globally. Here, we evaluated the status and histomorphological differentiation of TB lesions in 89 morbid cases of wild animals (bovids, cervids, carnivores, non-human primates, and pachyderms) in India. Histomorphological and molecular studies were done using Ziehl-Neelsen (ZN) staining, immunohistochemistry, and polymerase chain reaction (PCR), whereas cultural isolation was performed on selected samples. A total of 32 (35.95%) cases were confirmed as TB, comprising of 12 carnivores, 09 bovids, 06 cervids, 04 non-human primates, and a pachyderm. The TB lesions in the lungs, liver, and lymph nodes varied from the large-sized caseous nodules filled with dry cheesy material in bovids and cervids to variable-sized cavitations containing liquefied caseum in carnivores' lungs. The lungs, livers, and spleens of non-human primates exhibited small to medium-sized nodules. Histologically, lesions were divided into four categories (Types I, II, III, and IV) based on the extent of necrosis, the presence of mineralization, giant cells, and fibrous encapsulation. Extensive caseous necrosis with calcification, abundant giant cells, and thick fibroblastic encapsulation were consistent findings in the lungs, livers, and lymph nodes of bovids and cervids, whereas airway impaction with cellular exudate containing a teeming number of acid-fast bacilli and, at times, alveolar rupture leading to cavity formation was present in the lungs of carnivores. Absence of calcification and fibrous encapsulation was recorded in lungs of non-human primates. Immunohistochemical labelling with anti-early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) antibodies showed mild, moderate, and intense positive reactions in type II and III, type I, and type IV granulomatous lesions, respectively. Molecular detection by PCR revealed Mycobacterium tuberculosis (12 carnivores, 02 non-human primates and 01 pachyderm), M. bovis (02 cervids and 01 bovid) and M. orygis (02 cervids and 01 bovid). Cultural isolation confirmed M. tuberculosis in 03 carnivores and M. orygis in 02 cervids and 01 bovid. Our findings imply that TB is quite prevalent in the wildlife of India and there are considerable differences in the histomorphological lesions induced by distinct Mycobacterium species in different wild animals. The circulation of TB organisms in wild animals warrants a strict surveillance programme to identify the carrier status of these animals so that effective TB control strategies can be formulated to prevent spillover and spillback incidences at the human-livestock-wildlife interface.

Molecular Identification of Yersinia enterocolitica Isolated from Different Sources in Erbil City.

Yersinia enterocolitica ranks third among the pathogens that often cause the digestive disorder. It is transmitted to humans through food materials, especially contaminated meats. This research aimed to survey the frequency of Yersinia enterocolitica in sheep local products, especially meat, in Erbil. To carry out this study, 500 samples of raw milk, soft cheese, ice cream, and meat were collected by random sampling from different shops in Erbil City of Iraq. The samples were divided into four groups, including raw milk, soft cheese, ice cream, and meat. Many microbiological tests (culture, & staining, biochemical Tests, Vitek 2, and species-specific Polymerase Chain Reaction (PCR) amplicon for 16S RNA gene) were performed. Results showed that 5.1% of all of the studied samples were contaminated with Yersinia enterocolitica. Analysis of the results showed that meat is more contaminated than other samples. Also, the sequenced DNA evolutionary phylogeny tree of Yersinia enterocolitica isolates showed that all bacteria originated from the same genus and species. Therefore, it is recommended to pay special attention to this issue in order to avoid health and economic risks.

TRAX Provides Neuroprotection for Huntington's Disease Via Modulating a Novel Subset of MicroRNAs.

Huntington's disease (HD) is a neurodegenerative disease caused by CAG-repeat expansions (>36) in exon 1 of HTT, which dysregulates multiple cellular machineries. Translin-associated protein X (TRAX) is a scaffold protein with diverse functions, including suppressing the microRNA (miRNA)-mediated silencing by degrading pre-miRNA. To date, the role of TRAX in neurodegenerative diseases remains unknown. We delineated the role of TRAX upregulation during HD progression. Expression of TRAX in the brains of humans and three mouse models with HD were analyzed by immunohistochemistry staining, western blot, and quantitative reverse transcription-polymerase chain reaction. Adeno-associated viruses harboring TRAX short hairpin RNA were intrastriatally injected into HD mice to downregulate TRAX. HD-like symptoms were analyzed by behavioral and biochemical assessments. The miRNA-sequencing and RNA-sequencing analyses were used to identify the TRAX- regulated miRNA-messenger RNA (mRNA) axis during HD progression. The identified gene targets were validated biochemically in mouse and human striatal cells. We discovered that TRAX was upregulated in the brains of HD patients and three HD mouse models. Downregulation of TRAX enhanced 83 miRNAs (including miR-330-3p, miR-496a-3p) and subsequently changed the corresponding mRNA networks critical for HD pathogenesis (eg, DARPP-32 and brain-derived neurotrophic factor). Disruption of the TRAX-mediated miRNA-mRNA axis accelerated the progression of HD-like symptoms, including the degeneration of motor function, accumulation of mHTT aggregates, and shortened neurite outgrowth. We demonstrated that TRAX upregulation is authentic and protective in HD. Our study provides a novel layer of regulation for HD pathogenesis and may lead to the development of new therapeutic strategies for HD. © 2022 International Parkinson and Movement Disorder Society.

Lung-brain axis: Metabolomics and pathological changes in lungs and brain of respiratory syncytial virus-infected mice.

The lung-brain axis is an emerging area of study that got its basis from the gut-brain axis biological pathway. Using Respiratory Synctial Virus (RSV) as the model of respiratory viral pathogen, this study aims to establish some biological pathways. After establishing the mice model, the inflammation in lung and brain were assayed using Hematoxylin-eosin staining, indirect immunofluorescence (IFA), and quantitative reverse-transcription polymerase chain reaction. The biological pathways between lung and brain were detected through metabolomics analysis. In lung, RSV infection promoted epithelial shedding and infiltration of inflammatory cells. Also, RSV immunofluorescence and titerss were significantly increased. Moreover, interleukin (IL)-1, IL-6 and tumor necrosis factor-α (TNF-α) were also significantly increased after RSV infection. In brain, the cell structure of hippocampal CA1 area was loose and disordered. Inflammatory cytokines IL-6 and IL-1β expression in the brain also increased, however, TNF-α expression showed no differences among the control and RSV group. We observed an increased expression of microglia biomarker IBA-1 and decreased neuronal biomarker NeuN. In addition, RSV mRNA expression levels were also increased in the brains. 15 metabolites were found upregulated in the RSV group including nerve-injuring metabolite glutaric acid, hydroxyglutaric acid and Spermine. ɑ-Estradiol increased significantly while normorphine decreased significantly at Day 7 of infection among the RSV group. This study established a mouse model for exploring the pathological changes in lungs and brains. There are many biological pathways between lung and brain, including direct translocation of RSV and metabolite pathway.

Research on the osteogenesis and biosafety of ECM–Loaded 3D–Printed Gel/SA/58sBG scaffolds

Employing scaffolds containing cell–derived extracellular matrix (ECM) as an alternative strategy for the regeneration of bone defects has shown prominent advantages. Here, gelatin (Gel), sodium alginate (SA) and 58s bioactive glass (58sBG) were incorporated into deionized water to form ink, which was further fabricated into composite scaffolds by the 3D printing technique. Then, rat aortic endothelial cells (RAOECs) or rat bone mesenchymal stem cells (RBMSCs) were seeded on the scaffolds. After decellularization, two kinds of ECM–loaded scaffolds (RAOECs–ECM scaffold and RBMSCs–ECM scaffold) were obtained. The morphological characteristics of the scaffolds were assessed meticulously by scanning electron microscopy (SEM). In addition, the effects of scaffolds on the proliferation, adhesion, and osteogenic and angiogenic differentiation of RBMSCs were evaluated by Calcein AM staining and reverse transcription polymerase chain reaction (RT–PCR). In vivo, full–thickness bone defects with a diameter of 5 mm were made in the mandibles of Sprague–Dawley (SD) rats to assess the bone regeneration ability and biosafety of the scaffolds at 4, 8 and 16 weeks. The osteogenic and angiogenic potential of the scaffolds were investigated by microcomputed tomography (Micro–CT) and histological analysis. The biosafety of the scaffolds was evaluated by blood biochemical indices and histological staining of the liver, kidney and cerebrum. The results showed that the ECM–loaded scaffolds were successfully prepared, exhibiting interconnected pores and a gel–like ECM distributed on their surfaces. Consistently, in vitro experiments demonstrated that the scaffolds displayed favourable cytocompatibility. In vitro osteogenic differentiation studies showed that scaffolds coated with ECM could significantly increase the expression of osteogenic and angiogenic genes. In addition, the results from mandibular defect repair in vivo revealed that the ECM–loaded scaffolds effectively promoted the healing of bone defects when compared to the pure scaffold. Overall, these findings demonstrate that both RAOECs–ECM scaffold and RBMSCs–ECM scaffold can greatly enhance bone formation with good biocompatibility and thus have potential for clinical application in bone regeneration.

Neuroprotective effects of human umbilical cord mesenchymal stromal cells in PD mice via centrally and peripherally suppressing NLRP3 inflammasome-mediated inflammatory responses

Mesenchymal stromal cells (MSCs) exhibit beneficial anti-inflammatory effects against Parkinson’s disease (PD) via immunomodulatory actions. However, the underlying molecular mechanism remains unclear. This study aimed to investigate the potential neuroprotective effects of MSCs and the possible mechanisms involved by infusing human umbilical cord MSCs (hMSCs) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mature male C57BL/6 mice. Subsequently, the striatal content of dopamine (DA) and its metabolites, tyrosine hydroxylase (TH)-positive neurons and activated microglia, circulating inflammatory cytokines, gene expression of cytokines, and NOD-like receptor protein 3 (NLRP3) inflammasome molecules were measured using high-performance liquid chromatography, flow cytometry, immunohistochemistry, immunofluorescent staining, and real-time polymerase chain reaction assays, respectively. Infused hMSCs markedly ameliorated the reduction in striatal DA and loss of TH-positive neurons in the substantia nigra of PD mice. The MPTP-induced activation of microglia and increase in tumor necrosis factor-α and interleukin-1β mRNA expression in the striatum were also attenuated by hMSCs. Furthermore, hMSCs completely reversed the elevated pro-inflammatory cytokine levels in the serum and mRNA expression of cytokines in the peripheral organs of PD mice. Moreover, hMSCs significantly inhibited the expression of caspase-1 and NLRP3 of the NLRP3 inflammasome in both the central and peripheral organs at mRNA level. These data suggest that hMSCs protect dopaminergic neurons in PD mice by suppressing both the central and peripheral inflammatory responses, probably by inhibiting the NLRP3 inflammasome. However, the animals in our study only received several MSC infusions, and the effects of infused MSCs over extended periods on the NLRP3 inflammasome need to be investigated in future studies.

Conditional knockout of ASK1 in microglia/macrophages attenuates epileptic seizures and long-term neurobehavioural comorbidities by modulating the inflammatory responses of microglia/macrophages

BackgroundApoptosis signal-regulating kinase 1 (ASK1) not only causes neuronal programmed cell death via the mitochondrial pathway but also is an essential component of the signalling cascade during microglial activation. We hypothesize that ASK1 selective deletion modulates inflammatory responses in microglia/macrophages(Mi/Mϕ) and attenuates seizure severity and long-term cognitive impairments in an epileptic mouse model.MethodsMi/Mϕ-specific ASK1 conditional knockout (ASK1 cKO) mice were obtained for experiments by mating ASK1flox/flox mice with CX3CR1creER mice with tamoxifen induction. Epileptic seizures were induced by intrahippocampal injection of kainic acid (KA). ASK1 expression and distribution were detected by western blotting and immunofluorescence staining. Seizures were monitored for 24 h per day with video recordings. Cognition, social and stress related activities were assessed with the Y maze test and the three-chamber social novelty preference test. The heterogeneous Mi/Mϕ status and inflammatory profiles were assessed with immunofluorescence staining and real-time polymerase chain reaction (q-PCR). Immunofluorescence staining was used to detect the proportion of Mi/Mϕ in contact with apoptotic neurons, as well as neuronal damage.ResultsASK1 was highly expressed in Mi/Mϕ during the acute phase of epilepsy. Conditional knockout of ASK1 in Mi/Mϕ markedly reduced the frequency of seizures in the acute phase and the frequency of spontaneous recurrent seizures (SRSs) in the chronic phase. In addition, ASK1 conditional knockout mice displayed long-term neurobehavioral improvements during the Y maze test and the three-chamber social novelty preference test. ASK1 selective knockout mitigated neuroinflammation, as evidenced by lower levels of Iba1+/CD16+ proinflammatory Mi/Mϕ. Conditional knockout of ASK1 increased Mi/Mϕ proportion in contact with apoptotic neurons. Neuronal loss was partially restored by ASK1 selective knockout.ConclusionConditional knockout of ASK1 in Mi/Mϕ reduced seizure severity, neurobehavioral impairments, and histological damage, at least via inhibiting proinflammatory microglia/macrophages responses. ASK1 in microglia/macrophages is a potential therapeutic target for inflammatory responses in epilepsy.

Current Microbial Isolates and Their Antimicrobial Susceptibility Pattern from Wound Infection in a Tertiary Care Hospital

Background: Wound infection is one of the leading cause of mortality and morbidity worldwide. Objectives: To identify the current microbial isolates and their antimicrobial susceptibility pattern from wound infection in a tertiary care hospital in Dhaka city. Materials & Methods: This retrospective study was conducted in the department of Microbiology at Holy Family Red Crescent Medical College Hospital, Dhaka from July 2019 to December 2020 for a period of one and half years. Written consent was taken from corresponding authority. Microsoft Excel software was used for data analysis. A total 134 wound swab / pus were collected from the patients who were visited in outpatient department and were admitted at inpatient department with suspected wound infection. All samples were processed aerobically on Blood agar, MacConkeys agar media and incubated at 370C for 24 hours. Organisms were identified by standard procedures including colony characters, Gram staining and biochemical reactions. Antimicrobial susceptibility testing of all the isolates were performed by Kirby Bauer’s disc diffusion method. Results: A total 134 samples were analyzed. Of them, predominant populations were male 76(56.72%) and remaining were female 58(43.28%). Out of 134 samples 102 (76.11%) was culture positive. Culture positive was observed higher in inpatient department (IPD) 80 (78.43%) than outpatient department (OPD) 22 (21.57%). Majority of isolates were Gram negative bacteria. Among them predominant bacteria was E. coli 26(25.49%) followed by Klebsiella spp. 24(23.53%), Pseudomonas spp. 15(14.71%) & Proteus spp. 04(3.92%). Among the Gram - positive isolates 30(29.41%) was Staph. aureus. E. coli showed higher sensitivity to meropenem 88.46% & gentamicin 80.76%. Other drugs showed sensitivity to amikacin 65.38%, amoxicillin/clavulenic acid 53.84%, tetracycline 46.15%, ceftriaxone & ceftazidime 42.30%, trimethoprime/ sulfamethoxazole 34.61% & cefuroxime 23.07%. In Klebsiella spp. meropenem showed higher sensitivity 79.16% & gentamycin 66.66%. Other drugs like amikacin showed 58.33%, trimethoprime/ sulfamethoxazole 45.83%, ceftazidime & ciprofloxacin 20.83% sensitivity. Low sensitivity 16.66% to amoxicillin/clavulanic acid, cefuroxime, ceftriaxone, & tetracycline was found. Proteus spp. showed 75% sensitivity to meropenem. Cefuroxime, ceftriaxone, ciprofloxacin, ceftazidime, gentamycin, amikacin, trimethoprime/ sulfamethoxazole showed very low sensitivity 25% & amoxycillin/clavulanic acid showed 100% resistance. Pseudomonas spp. showed highest sensitivity 83.33% to piperacillin tazobactum, amikacin & imipenem, 77.77% to meropenem, 66.66% to gentamycin, 61.11% to ciprofloxacin, 50% to ceftazidime & 33.33% to cefepime. All the isolate of Staph. aureus was sensitive to amikacin 90%, amoxyclav 80%, cloxacillin 70%, ciprofloxacin 70%, gentamicin 63.33% & azythromycin showed lower sensitivity 43.33%. Conclusion: Most of the injectable antibiotics showed higher sensitivity which is an alarming sign for the clinician to treat wound infection. Periodic review of the bacteriological profile and antibiotic sensitivity pattern is highly essential. Antibiotic policy & infection control program should be included in every hospital to reduce this drug resistance. J Shaheed Suhrawardy Med Coll 2021; 13(1): 80-85

Melatonin enhances autologous adipose-derived stem cells to improve mouse ovarian function in relation to the SIRT6/NF-κB pathway

BackgroundPremature ovarian insufficiency (POI) is the main cause of female infertility. Adipose-derived stem cells (ADSCs) are ideal candidates for the treatment of POI. However, some deficient biological characteristics of ADSCs limit their utility. This study investigated whether melatonin (MLT)-pretreated autologous ADSCs were superior to ADSCs alone in the treatment of the POI mouse model.MethodsAutologous ADSCs were isolated and cultured in MLT-containing medium. Surface markers of ADSCs were detected by flow cytometry. To determine the effect of MLT on ADSCs, CCK-8 assay was used to detect ADSCs proliferation and enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of cytokines. The POI model was established by intraperitoneal injection of cyclophosphamide and busulfan. Then, MLT-pretreated autologous ADSCs were transplanted into mice by intraovarian injection. After 7 days of treatment, ovarian morphology, follicle counts, and sex hormones levels were evaluated by hematoxylin and eosin (H&E) staining and ELISA, and the recovery of fertility was also observed. The expressions of SIRT6 and NF-κB were detected by immunohistochemical (IHC) staining and quantitative real-time polymerase chain reaction (qRT-PCR).ResultsFlow cytometry showed that autologous ADSCs expressed CD90 (99.7%) and CD29 (97.5%). MLT can not only promote the proliferation of ADSCs but also boost their secretory function, especially when ADSCs were pretreated with 5 µM MLT for 3 days, improving the interference effect. After transplantation of autologous ADSCs pretreated with 5 µM MLT, the serum hormone levels and reproductive function were significantly recovered, and the mean counts of primordial follicle increased. At the same time, the expression of SIRT6 was remarkably increased and the expression of NF-κB was significantly decreased in this group.ConclusionsMLT enhances several effects of ADSCs in restoring hormone levels, mean primordial follicle counts, and reproductive capacity in POI mice. Meanwhile, our results suggest that the SIRT6/NF-κB signal pathway may be the potential therapeutic mechanism for ADSCs to treat POI.

Fibroblast-derived exosomal miRNA-133 promotes cardiomyocyte-like differentiation

ObjectiveTo investigate the role of exosomal miRNA-133 secreted by cardiac fibroblasts (CFs) in promoting cardiomyocyte differentiation. MethodsNeonatal rat CFs were cultured in vitro, and the cultured CFs were divided into three groups as follows: induction, miRNA-133 high expression, and miRNA-133 inhibition. miRNA-133 was transfected into CFs with lentivirus as a vector. CFs were transfected with the miRNA-133 inhibitor, and the markers of cardiomyocyte were detected through immunofluorescence staining, Western blotting, and real-time quantitative polymerase chain reaction (qRT-PCR) at 3, 8, and 14 days, respectively. The expression levels of cardiac troponin T (cTnT) and cardiac actin (α-actin) were determined, and qRT-PCR was used to detect the expression of miRNA-133 in the fibroblast exosomes. ResultsCFs subjected to immunofluorescence staining expressed vimentin and discoid domain receptor 2. The exosomes secreted by CFs were observed as small vesicles of 30–100 nm via transmission electron microscopy, and Western blotting was used to detect exosome-specific protein CD63 and CD9 expression. The expression levels of cTnT, α-actin, and exosomal miRNA-133 secreted into the supernatant of the miRNA-133 high-expression group increased gradually at different time points and reached the highest level at 14 days. The expression levels of cTnT, α-actin, and exosome miRNA-133 in the miRNA-133 inhibition group were the lowest. ConclusionThe exosomal miRNA-133, which is derived from CFs, can promote the differentiation of fibroblasts into cardiomyocyte-like cells.

Inhibition of autophagy by 3-methyladenine promotes migration and invasion of colon cancer cells through epithelial mesenchymal transformation

BackgroundColon cancer is the third leading cause of tumor-related deaths in the world. Inhibition of autophagy in the treatment of malignant tumors has attracted extensive attention. However, the association between inhibition of autophagy by 3-methyladenine (3-MA) and epithelial mesenchymal transformation (EMT) in colon cancer cells has not yet been fully elucidated.MethodsIn this study, colon cancer cell lines (LOVO and SW620) were treated with 3-MA. Wound healing assays and transwell assays were used to detect the effect of inhibition of autophagy on the migration and invasion of colon cancer cells. The expression of EMT-associated markers, Twist1, E-cadherin, and vimentin, in colon cancer cells with and without 3-MA treatment was detected by Western blotting, immunohistochemistry, immunofluorescence staining, and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR).ResultsOur data showed that inhibition of autophagy by 3-MA significantly enhanced the migration and invasion of colon cancer cells. At the molecular level, inhibition of autophagy upregulated the expression of Twist1 and vimentin, downregulated the expression of E-cadherin, and induced the EMT of colon cancer cells.ConclusionsInhibition of autophagy by 3-MA upregulated the expression of Twist1 in colon cancer cells and promoted cancer cell migration and invasion through EMT. Inhibition of autophagy may have adverse effects on colon cancer.

Investigation of Al-toxicity tolerance in tef (Eragrostis tef) under hydroponic system using root growth measurement and haematoxylin staining methods

Tef is the most widely grown and consumed crop in Ethiopia. The crop is currently gaining growing popularity worldwide as nutritious and gluten free cereal crop. Al-toxicity is one of the factors that limit expansion of the crop worldwide. The aims of this study were to adapt hydroponic system as a phenotyping platform for Al-tolerance studies in tef, to identify appropriate concentration of Al3+ for Al-tolerance screening in tef, and to appraise the use of haematoxylin staining for visual assessment of Al-tolerance in tef. A tolerant and a sensitive genotypes were used to identify appropriate Al concentration. The identified Al concentration was used to evaluate reaction of 28 tef genotypes to Al-toxicity. Root and shoot measurements under Al-treated and control solution were used to compute the relative tolerance indexes. Reaction of the tolerant and the sensitive genotypes to haematoxylin staining was assessed by recording intensity of colour development. The result indicate that, among the five levels of AlK(SO4)2.12H2O (0-550 µM), the 150 µM concentration was found to be an appropriate level to discriminate between sensitive and tolerant genotypes of tef. This concentration adequately differentiated 28 tef genotypes with varied sensitivity to Al-toxicity. The visual assessment of selected tolerant and sensitive genotypes treated with 0, 150 and 250 µM AlK(SO4)2 .12H2O showed a differential reaction to haematoxylin staining consistent with the root growth measurement methods. The tolerant E. curvula showed no staining reaction across all the Al levels. The local Al-tolerant tef landrace, Dabo banja, showed a slightly purplish stain only at the highest level of Al. The two sensitive genotypes (E. Pilossa and Holeta Key), however, showed deep purple staining at concentrations of 150 and 250 µM AlK(SO4)2.12H2O. To the best of the authors’ knowledge, this study is the first to report the use of root measurement and root staining methods in evaluation of tef for tolerance to Al-toxicity using hydroponics platform.

Bacteriological Profile and Antibiotic Susceptibility of Blood Culture Isolates in Intensive Care Units of a Tertiary Care Hospital

Intensive Care Units infections have been found to be highest amidst the nosocomial infections. Sepsis results in increased morbidity and mortality rates. In order to decrease mortality and morbidity rates associated with sepsis, intensivists should have a keen knowledge of the existing bacteriological flora and their antibiotic susceptibility pattern. In this study, we aim to determine the prevalence of current bacteriological profiles in blood cultures, along with their antibiogram from Intensive Care Unit patients. This project was a retrospective cross-sectional study carried out for the duration of one year from December 2018 to 2019 in the Central lab, Microbiology department at tertiary care hospital, from patients suspicious of some blood stream infections like sepsis or other risk factors for it. From ICU, the number of blood samples received was 1440 in our Central Microbiology laboratory. All the samples received in the microbiology central lab were processed, and laboratory data, including bacteriological profile and antimicrobial susceptibilities were analysed. From ICU, the number of blood samples totally received were 1440 in our microbiology laboratory. Identification of isolates was done by colony morphology, Gram stain and standard biochemical reactions. Antibiotic susceptibility pattern was conferred by Clinical and Laboratory Standards Institute guidelines. Total number of positive cultures present in the study was 156(10.8%). Among these culture growth organisms, 82 were Gram positive (55.4%) isolates and 74 were Gram negative (50%). The most common isolate was Coagulase negative Staphylococcus trailed by Escherichia coli and Staphylococcus aureus, Acinetobacter species and Pseudomonas aeruguinosa, Klebsiella pneumoniae and Enterococcus species. Majority of the Gram positive isolates were susceptible to Linezolid, vancomycin and clindamycin. All the Gram negative isolates were susceptible to Carbapenems ,with susceptibility rate of 97% for E. coli, 93% for Acinetobacter, 93% for Pseudomonas aeruguinosa and almost all these isolates showed 100% susceptibility to Colistin and Polymyxin-B.

Ectoderm-derived frontal bone mesenchymal stem cells promote traumatic brain injury recovery by alleviating neuroinflammation and glutamate excitotoxicity partially via FGF1

BackgroundTraumatic brain injury (TBI) leads to cell and tissue impairment, as well as functional deficits. Stem cells promote structural and functional recovery and thus are considered as a promising therapy for various nerve injuries. Here, we aimed to investigate the role of ectoderm-derived frontal bone mesenchymal stem cells (FbMSCs) in promoting cerebral repair and functional recovery in a murine TBI model.MethodsA murine TBI model was established by injuring C57BL/6 N mice with moderate-controlled cortical impact to evaluate the extent of brain damage and behavioral deficits. Ectoderm-derived FbMSCs were isolated from the frontal bone and their characteristics were assessed using multiple differentiation assays, flow cytometry and microarray analysis. Brain repairment and functional recovery were analyzed at different days post-injury with or without FbMSC application. Behavioral tests were performed to assess learning and memory improvements. RNA sequencing analysis, immunofluorescence staining, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were used to examine inflammation reaction and neural regeneration. In vitro co-culture analysis and quantification of glutamate transportation were carried out to explore the possible mechanism of neurogenesis and functional recovery promoted by FbMSCs.ResultsEctoderm-derived FbMSCs showed fibroblast like morphology and osteogenic differentiation capacity. FbMSCs were CD105, CD29 positive and CD45, CD31 negative. Different from mesoderm-derived MSCs, FbMSCs expressed the ectoderm-specific transcription factor Tfap2β. TBI mice showed impaired learning and memory deficits. Microglia and astrocyte activation, as well as neural damage, were significantly increased post-injury. FbMSC application ameliorated the behavioral deficits of TBI mice and promoted neural regeneration. RNA sequencing analysis showed that signal pathways related to inflammation decreased, whereas those related to neural activation increased. Immunofluorescence staining and qRT-PCR data revealed that microglial activation and astrocyte polarization to the A1 phenotype were suppressed by FbMSC application. In addition, FGF1 secreted from FbMSCs enhanced glutamate transportation by astrocytes and alleviated the cytotoxic effect of excessive glutamate on neurons.ConclusionsEctoderm-derived FbMSC application significantly alleviated neuroinflammation, brain injury, and excitatory toxicity to neurons, improved cognition and behavioral deficits in TBI mice. Therefore, ectoderm-derived FbMSCs could be ideal therapeutic candidates for TBI which mostly affect cells from the same embryonic origins as FbMSCs.

Roles of immune cells in the concurrence of Echinococcus granulosus sensu lato infection and hepatocellular carcinoma

Immune cells are pivotal players in the immune responses against both parasitic infection and malignancies. Substantial evidence demonstrated that there may exist possible relationship between echinococcus granulus sensu lato (E. granulosus s.l.) infection and hepatocellular carcinoma (HCC) development. Thus, this study aimed to observe crucial roles of immune cells in the formation of subcutaneous lesions after transplanting HepG2 cell lines with or without E. granulosus s.l. protoscoleces (PSCs). HepG2 cell lines were subcutaneously injected into nude mice in the control group. In the co-transplantation group, HepG2 cells were subcutaneously co-injected with high dosage of E. granulosus s.l. PSCs. From the 25th day of transplantation, volume of subcutaneous lesions was measured every four days, which were removed at the 37th day for further studies. Basic pathological and functional changes were observed. Moreover, expression of Ki67, Bcl-2, Caspase3, α-smooth muscle actin (α-SMA), T cell markers (CD3, CD4, CD8), PD1/PD-L1, nature killer (NK) cell markers (CD16, CD56) were further detected by immunohistochemical staining and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Subcutaneous lesions were gradually increased in volume and there occurred pathologically heterogeneous tumor cells, which were more significant in the co-transplantation group. Compared to the control group, expression of proliferation markers Ki67 and Bcl-2 was at higher levels in the co-transplantation group. Reversely, apoptotic marker Caspase3 was highly detected in the control group, suggesting promoting effects of E. granulosus s.l. PSCs on HCC development. Interestingly, subcutaneous lesions of the co-transplantation group were more functional in synthesizing and storing glycogen. Collagen and α-SMA+ cells were also at higher levels in the co-transplantation group than those in the control group. Most importantly, co-transplantation of HepG2 cells with E. granulosus s.l. PSCs led to significant increase in the expression of T cell markers, PD1/PD-L1 and NK cells markers. E. granulosus s.l. may have promoting effects on HCC development, which was closely associated with the immune responses of T cells and NK cells.

GDF11 inhibits abnormal adipogenesis of condylar chondrocytes in temporomandibular joint osteoarthritis.

AimsAbnormal lipid metabolism is involved in the development of osteoarthritis (OA). Growth differentiation factor 11 (GDF11) is crucial in inhibiting the differentiation of bone marrow mesenchymal stem cells into adipocytes. However, whether GDF11 participates in the abnormal adipogenesis of chondrocytes in OA cartilage is still unclear.MethodsSix-week-old female mice were subjected to unilateral anterior crossbite (UAC) to induce OA in the temporomandibular joint (TMJ). Histochemical staining, immunohistochemical staining (IHC), and quantitative real-time polymerase chain reaction (qRT-PCR) were performed. Primary condylar chondrocytes of rats were stimulated with fluid flow shear stress (FFSS) and collected for oil red staining, immunofluorescence staining, qRT-PCR, and immunoprecipitation analysis.ResultsAbnormal adipogenesis, characterized by increased expression of CCAAT/enhancer-binding protein α (CEBPα), fatty acid binding protein 4 (FABP4), Perilipin1, Adiponectin (AdipoQ), and peroxisome proliferator-activated receptor γ (PPARγ), was enhanced in the degenerative cartilage of TMJ OA in UAC mice, accompanied by decreased expression of GDF11. After FFSS stimulation, there were fat droplets in the cytoplasm of cultured cells with increased expression of PPARγ, CEBPα, FABP4, Perilipin1, and AdipoQ and decreased expression of GDF11. Exogenous GDF11 inhibited increased lipid droplets and expression of AdipoQ, CEBPα, and FABP4 induced by FFSS stimulation. GDF11 did not affect the change in PPARγ expression under FFSS, but promoted its post-translational modification by small ubiquitin-related modifier (SUMOylation). Local injection of GDF11 alleviated TMJ OA-related cartilage degeneration and abnormal adipogenesis in UAC mice.ConclusionAbnormal adipogenesis of chondrocytes and decreased GDF11 expression were observed in degenerative cartilage of TMJ OA. GDF11 supplementation effectively inhibits the adipogenesis of chondrocytes and thus alleviates TMJ condylar cartilage degeneration. GDF11 may inhibit the abnormal adipogenesis of chondrocytes by affecting the SUMOylation of PPARγ.Cite this article: Bone Joint Res 2022;11(7):453–464.

Etiology of cervicitis: Are there new agents in play?

Considering the changing causative and resistance pattern of agents implicated in sexually transmitted infections (STIs), etiological diagnosis is imperative, especially in countries practicing syndromic management. This study was designed to identify etiological agents associated with cervicitis and to analyze their association with clinical and behavioral profile. Female STI clinic attendees presenting with cervico-vaginal discharge were examined for the presence of cervicitis. Endocervical swabs were collected for gram staining and real-time polymerase chain reaction was performed for various bacterial and viral STI agents in patients presenting with cervical discharge. A vaginal swab was also evaluated for bacterial vaginosis by Nugent's criteria. Of 64 patients with vaginal discharge, 26.6% and 12.5% patients complained of genital itching and lower abdominal pain, respectively. Mean of 36.6 pus cells/hpf were observed, appreciably greater number in patients with Neisseria gonorrhoeae and Chlamydia trachomatis infections (P = 0.0063 and 0.0032, respectively). Pus cells were high (mean 68 pus cells/hpf) in patients with Ureaplasma urealyticum, though this may be attributed to coexisting N. gonorrhoeae. Agents isolated from endocervix were N. gonorrhoeae, 17 (26.6%), Trichomonas vaginalis, 4 (6.3%), HSV1 and C. trachomatis, 1 each (1.6%), HSV2, 9 (14.1%), U. urealyticum 5 (7.8%), Ureaplasma parvum 26 (40.6%), Mycoplasma genitalium (0%), and Mycoplasma hominis 11 (17.2%). Bacterial vaginosis was diagnosed in 14 (21.9%) patients. Multiple agents were isolated in 10 (two), 6 (three), 6 (four), and 1 (five) patients. Isolation of M. hominis and U. parvum was significantly associated with bacterial vaginosis (P = 0.04 and 0.003, respectively). Nonusage of condoms and mental stress predisposed to cervicitis. We concluded that there are changing etiological patterns of cervicitis. There is need to use tests that detect wider array of organisms, and can replace standard culture methods with molecular assays, to increase the ability to diagnose more number of organisms implicated in cervicitis.