Regulation of Clostridium tetani Neurotoxin Expression by Culture Conditions

Background: Ensuring consistency of tetanus neurotoxin (TeNT) production by Clostridium tetani could help to ensure consistent product quality in tetanus vaccine manufacturing, ultimately contributing to reduced animal testing. The aim of this study was to identify RNA signatures related to consistent TeNT production using standard and non-standard culture conditions. Methods: We applied RNA sequencing (RNA-Seq) to study C. tetani gene expression in small-scale batches under several culture conditions. Results: We identified 1381 time-dependent differentially expressed genes (DEGs) reflecting, among others, changes in growth rate and metabolism. Comparing non-standard versus standard culture conditions identified 82 condition-dependent DEGs, most of which were specific for one condition. The tetanus neurotoxin gene (tetX) was highly expressed but showed expression changes over time and between culture conditions. The tetX gene showed significant down-regulation at higher pH levels (pH 7.8), which was confirmed by the quantification data obtained with the recently validated targeted LC-MS/MS approach. Conclusions: Non-standard culture conditions lead to different gene expression responses. The tetX gene appears to be the best transcriptional biomarker for monitoring TeNT production as part of batch-to-batch consistency testing during tetanus vaccine manufacturing.

A vascularized 3D model of the human pancreatic islet for ex vivo study of immune cell-islet interaction

Insulin is an essential regulator of blood glucose homeostasis that is produced exclusively by β cells within the pancreatic islets of healthy individuals. In those affected by diabetes, immune inflammation, damage, and destruction of islet β cells leads to insulin deficiency and hyperglycemia. Current efforts to understand the mechanisms underlying β cell damage in diabetes rely on in vitro-cultured cadaveric islets. However, isolation of these islets involves removal of crucial matrix and vasculature that supports islets in the intact pancreas. Unsurprisingly, these islets demonstrate reduced functionality over time in standard culture conditions, thereby limiting their value for understanding native islet biology. Leveraging a novel, vascularized micro-organ (VMO) approach, we have recapitulated elements of the native pancreas by incorporating isolated human islets within a three-dimensional matrix nourished by living, perfusable blood vessels. Importantly, these islets show long-term viability and maintain robust glucose-stimulated insulin responses. Furthermore, vessel-mediated delivery of immune cells to these tissues provides a model to assess islet-immune cell interactions and subsequent islet killing -- key steps in type 1 diabetes pathogenesis. Together, these results establish the islet-VMO as a novel, ex vivo platform for studying human islet biology in both health and disease.

Seroprevalence of brucellosis and molecular characterization of Brucella spp. from slaughtered cattle in Rwanda

Bovine brucellosis is endemic in Rwanda, although, there is paucity of documented evidence about the disease in slaughtered cattle. A cross-sectional study was conducted in slaughtered cattle (n=300) to determine the seroprevalence of anti-Brucella antibodies using the Rose Bengal Test (RBT), and indirect enzyme-linked immunosorbent assay (i-ELISA). Corresponding tissues were cultured onto a modified Centro de Investigación y Tecnología Agroalimentaria (CITA) selective medium and analysed for Brucella spp. using the 16S-23S ribosomal interspacer region (ITS), AMOS, and Bruce-ladder PCR assays. The RBT seroprevalence was 20.7% (62/300), and 2.9% (8/300) with i-ELISA and 2.9% (8/300) using both tests in parallel. Brucella specific 16S-23S ribosomal DNA interspace region (ITS) PCR detected Brucella DNA in 5.6% (17/300; Brucella culture prevalence). AMOS-PCR assay identified mixed B. abortus and B. melitensis (n=3), B. abortus (n=3) and B. melitensis (n=5) while Bruce-ladder PCR also identified B. abortus (n=5) and B. melitensis (n=6). The gold standard culture method combined with PCR confirmation identified 5.6% Brucella cultures which is higher than the more sensitive seroprevalence of 2.9%. This emphasizes the need to validate the serological tests in Rwanda. The mixed infection caused by B. abortus and B. melitensis in slaughtered cattle indicates cross-infection and poses a risk of exposure potential to abattoir workers. It is essential to urgently strengthen the national bovine brucellosis control program through vaccination as well as test- and-slaughter.

Prevalence of Methicillin-Resistant Staphylococcus Species Among Filarial Lymphedema Patients in Ahanta West District of Ghana

BackgroundFilarial pathologies such as lymphedema may be associated with complications such as chronic non-healing wounds. Nonetheless, the role of bacterial population colonizing the lymphedematous legs has been posited to worsen the conditions of those living with the infection. These bacteria are usually composed of staphylococcal species partly because they are commensals. Thus, this present study sought to type the methicillin-resistant Staphylococcus aureus (MRSA) prevalence among individuals presenting with filarial lymphedema, particularly as MRSA tends to affect treatments options.MethodsWe recruited individuals (n = 321) with stages I–VII of lymphedema in a cross-sectional study in the Ahanta West district of the Western Region of Ghana. Swabs from lymphedematous limb ulcers, pus, and cutaneous surfaces were cultured using standard culture-based techniques. The culture isolates were later identified using Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry.ResultsA total of 192 Staphylococci species were isolated, with an overall prevalence of 39.7% (95% CI: 35%–44%; N = 483). S. hominis was the most prevalent species (23.95%), followed by S. haemolyticus (20.83%), S. epidermidis (15.10%), S. aureus (10.41%), and S. saprophyticus (9.32%). The remaining 20.34% were distributed among S. wanneri, S. sciuri, S. pasteuri, S. xylosus, S. simulans, S. cohnii, S. caprae, S. lugdunensis, and S. capitis. MRSA, containing mecA gene, was detected in 21 out of 31 Staphylococci isolates tested, with an overall prevalence of 68% (95% CI: 51%–84%). In addition, a virulent gene, Panton–Valentine leukocidin (PVL), which is usually associated with S. aureus, was detected in 20/31 (64.5%) S. aureus in the study.ConclusionThese results suggest that MRSA species may pose a challenge to the treatment of filarial lymphedema with antibiotics particularly, as doxycycline is currently being piloted in some endemic areas to treat the infection. Thus, intensive antimicrobial resistance surveillance should be conducted in endemic areas by health authorities to forestall the dilemma of multidrug resistance not only against lymphatic filariasis (LF) infection but other diseases.

Mammalian HEMK1 Methylates Glutamine Residue of the GGQ Motif of Mitochondrial Release Factors

Despite limited reports on glutamine methylation, methylated glutamine is found to be highly conserved in a "GGQ" motif in both prokaryotes and eukaryotes. In bacteria, glutamine methylation of peptide chain release factors 1/2 (RF1/2) by the enzyme PrmC is essential for translational termination and transcript recycling. Two PrmC homologs, HEMK1 and HEMK2, are found in mammals. In contrast to those of HEMK2, the biochemical properties and biological significance of HEMK1 remain largely unknown. In this study, we demonstrated that HEMK1 is an active methyltransferase for the glutamine residue of the GGQ motif of all four putative mitochondrial release factors (mtRFs)—MTRF1, MTRF1L, MRPL58, and MTRFR. In HEMK1-deficient HeLa cells, GGQ motif glutamine methylation was absent in all the mtRFs. We examined cell growth and mitochondrial properties, but disruption of the HEMK1 gene had no considerable impact on the overall cell growth, mtDNA copy number, and mitochondrial membrane potential. Furthermore, mitochondrial protein synthesis was not affected in HEMK1 KO cells. Our results suggest that HEMK1 mediates the GGQ methylation of all four mtRFs in human cells; however, this specific modification seems mostly dispensable in cell growth and mitochondrial protein homeostasis under standard culture conditions.

Abstract 14580: Methodological Challenges Conducting In Vitro Ischemia and Reperfusion: A Fundamental Problem in Cell Viability Determination

Background: Oxygen & Glucose Deprivation (OGD) and Reperfusion (OGD-R) is considered one of the best models for studying ischemia and reperfusion injury (IRI) in vitro. Cell viability (CV) experimentation can aid in estimating cell survival after IRI insults and potential benefits from therapeutic attempts. However, the different metabolites used in various CV assays may compromise the usefulness and validity of various CV tools. We conducted two independent cell viability assays (WST-8 & ATP) after OGD and OGD-R on neurons (NE) and astrocytes (AS). Methods: NE (HT-22) and AS (C8-D1A) were subjected to 6h OGD only and 6h OGD followed by 20h Reperfusion (OGD-R). OGD is based on the combination of chemical ischemia (standard culture media depleted of glucose and other energy sources) and severe hypoxia (O2 ~1.4 to 1.8%) for 6h, while 20h reperfusion consists of readdition of standard culture media and conditions (21% O2, 5% CO2, 37°C). CV was determined by WST-8 and ATP “CellTiter-Glo 2.0” assays. Results: By WST-8 assay, OGD decreased CV in NE (Fig. 1A, P value = 0.0121), but increased CV in AS (Fig. 1A, P value = 0.0079). OGD-R decreased CV in NE and AS (Fig. 2A, P value = 0.0010, 0.0146, respectively). By ATP assay, OGD decreased CV in NE (Fig. 1B, P value <0.0001) and AS (Fig. 1C, P value <0.0001). OGD-R also decreased CV in NE (Fig. 2B, P value <0.0001) and AS (Fig. 2C, P value = 0.0001). Conclusions: The current data reveals opposite CV results after OGD in AS when comparing WST-8 and ATP assays; OGD caused CV decrease in NE but increased in AS, which constitutes a paradoxical response based on previous literature. However, OGD-R caused similar injury patterns in NE and AS from WST-8 and ATP assays. It is unknown why OGD increased CV in AS through WST-8 after OGD, however, it can potentially occur due to AS hypermetabolism leading to increased NADPH+ production, which is the main substrate in this CV estimation. We conclude that CV should be carefully assessed with multiple, independent CV assays.

MICROBIAL PRODUCTION OF FERULIC ACID AND OPTIMIZATION OF THE MEDIUM PARAMETERS USING FRACTIONAL FACTORIAL DESIGN

Phenolics are widely distributed in plant kingdom and are therefore, an integral part of the diet, with significant amounts being reported in vegetables, fruits, and beverages. Various phenolic compounds have attracted the attention of food and medical scientists because of their fragrance, aroma, antioxidant, anti-inflammatory properties and ability to combat human diseases. Of these, Ferulic Acid (FA), a hydroxy cinnamic acid (related to trans-cinnamic acid), being natural, is of great demand in the food industry. As a component of lignin, FA is a precursor in the manufacture of other aromatic compounds. In our study, FA was produced using Lactobacillus spp. isolates and standard culture of Lactobacillus plantarum ATCC 8014. FA was extracted and partially characterized using Thin Layer Chromatography (TLC), Absorption maxima (λm) analysis and High Performance Liquid Chromatography (HPLC). Further, optimization of the fermentation medium was done using Factorial Fractional Design (FFD). Preliminary confirmation of the extracted FA was done using TLC, spectral analysis and purity assessed by HPLC. FA could be produced using Lactobacillus sp. and agro industrial waste viz., wheat bran, leading to a cost-effective protocol and product. Further, medium was optimized for the production of FA using FFD and it was observed that medium containing5.75% Wheat bran & 0.18% Tween 80 is optimum for the production of FA. The antimicrobial activity of FA was noteworthy against Aspergillus flavus and E. coli.

Fine-needle aspiration to improve diagnosis of melioidosis of the head and neck in children: a study from Sarawak, Malaysia

Background Melioidosis, the infection caused by Burkholderia pseudomallei, is associated with a high case fatality rate, due in part to difficulties in clinical recognition and diagnostic confirmation of the disease. Although head and neck involvement is common in children, specific disease manifestations differ between geographic regions. The aim of this study was to provide a detailed description of melioidosis of the head and neck among children in Sarawak, Malaysia, and determine if fine-needle aspiration of suspected head or neck lesions could improve melioidosis diagnosis. Methods We conducted a retrospective descriptive study of all children aged < 12 years with culture-confirmed melioidosis presenting with head and neck manifestations and admitted to Bintulu Hospital in Sarawak, Malaysia, from January 2011 until December 2020. Fine-needle aspiration of head and neck lesions suspected to be due to melioidosis with inoculation in blood culture bottles (FNA + BCB) was used from the beginning of 2016. Results Of 34 children with culture-confirmed melioidosis, 20 (59%) had an infection involving one or more sites in the head and neck. Of these, 17 (85%) were diagnosed in or after 2016. Cervical lymph nodes were the most common organ or site affected, involved in 19 (95%) children. Clinical presentations of B. pseudomallei lymph node infections were highly variable. Five (25%) children had salivary gland involvement. Lacrimal gland involvement (dacryocystitis) and skin or soft tissue infection (scalp abscess) were less frequent. B. pseudomallei was isolated from the head or neck using FNA + BCB in 15 (75%) children and by standard culture methods of direct plating of pus on agar following incision and drainage in only 2 (10%) children. B. pseudomallei was isolated from non-head or neck specimens or blood in 3 (15%) children. Conclusions Manifestations of pediatric head and neck melioidosis in Sarawak, Malaysia, differ from those of other regions. Fine-needle aspiration, mainly of affected cervical lymph nodes, facilitates B. pseudomallei detection and enables confirmation of melioidosis infections.

Induced Dryness Stress on Human Vaginal Epithelium: The Efficacy of a New Vaginal Gel

An experimental model of dryness on vaginal mucosa is proposed to assess the efficacy of a new vaginal gel (Respecta® Hydragel Ref 17031). The dryness model was induced on reconstituted human vaginal epithelium (HVE) by incubating the tissues in modified environmental conditions (R.H. < 50% and T = 40 °C) for 48 h. The products were applied on the ‘Dry’ HVE models for 24 h (series 48 h + 24 h) in standard culture conditions (37 °C 5% CO2). Their efficacy in counteracting vaginal dryness was assessed and compared to tissues treated with saline solution and cultured in standard culture conditions (negative control) and to untreated tissues incubated in dryness conditions for 48 h and then recovered after 24 h in standard culture conditions (positive control). The products’ efficacy was quantified by measuring the following parameters: (1) water flux and direct moisturization by AQP3 immunohistochemical staining, and (2) maintenance of moisturization and elasticity of the mucosa by hyaluronic acid (CD44) immunofluorescence staining. Respecta® Hydragel demonstrated efficacy in regulating the water flux by inducing AQP3 expression thus determining a positive water balance within the vaginal epithelium. It induced a remodelling of the epithelium morphology with restored trophism compared to the dry HVE control. Furthermore, it demonstrated a significant increase of the expression of CD44, related to hyaluronic acid (HA) distribution in the extracellular matrix. HA has the ability to act on the cellular matrix composition and its renewal compared to the dry HVE control. Through these mechanisms it induces a deep hydration and elasticity of the vaginal mucosa.

Novel culture media enhances mononuclear cells from patients with chronic limb-threatening ischemia to increase vasculogenesis and anti-inflammatory effect

Background Quality and Quantity culture media (QQ culture media) was reported to enhance vasculogenesis and angiogenesis function of mononuclear cells (MNCs) from healthy volunteers. In this study, MNCs from chronic limb-threatening ischemia (CLTI) patients were cultured in QQ culture media, and then investigated for angiogenesis-related phenotype and function. Methods Patients aged ≥ 18 years with CLTI caused by atherosclerosis of the lower extremities were prospectively recruited at Siriraj Hospital (Bangkok, Thailand) during July 2017–December 2018. Peripheral blood mononuclear cells (PBMNCs) were isolated from peripheral blood. PBMNCs were cultured in either QQ culture media or standard culture media. The number of CD34+CD133+ cells, CD206+ cells, CD4+CD25+CD127+ cells, colony formation assay, and human umbilical vein endothelial cell (HUVEC) tube formation assay in MNCs were compared between those cultured in QQ culture media and those cultured in standard culture media. Results Thirty-nine patients were included with a mean age of 69 ± 11 years. Diabetes mellitus was found in 25 (64%) patients. The percentage of CD34+CD133+ progenitor cells in MNCs cultured in QQ culture media and in MNCs cultured in standard culture media was 4.91 ± 5.30% and 0.40 ± 0.46%, respectively (p < 0.0001). The percentage of CD206+ cells in MNCs cultured in QQ culture media and in MNCs cultured in standard culture media was 19.31 ± 11.42% and 4.40 ± 2.54%, respectively (p < 0.0001). The percentage of inactive population of T regulatory cells (CD4+CD25+CD127+ cells) in MNCs cultured in standard culture media and in MNCs cultured in QQ culture media was 14.5 ± 10.68% and 1.84 ± 1.37%, respectively (p < 0.0001). The total number of colony-forming units from MNCs cultured in QQ culture media and in MNCs cultured in standard culture media was 8.86 ± 8.35 of 2 × 105 cells/dish, and 0.58 ± 1.05 of 2 × 105 cells/dish, respectively (p < 0.0001). The mean intensity of Dil-Ac-LDL uptake that incorporated into the HUVEC forming tube was 1.37 ± 0.88 in MNCs cultured in QQ culture media, and 0.78 ± 0.41 in MNCs cultured in standard culture media. (p < 0.0003). Conclusions MNCs from CLTI patients that were cultured in QQ culture media had a significantly higher number of CD34+CD133+ cells and anti-inflammatory cells, and higher angiogenesis-related function compared to MNCs cultured in standard culture media.