Impact of COVID-19 infections and vaccination on menstrual cycle symptoms in the south of Jordan: a cross-sectional study.

BACKGROUND: Several recent studies have highlighted the need for more evaluation of the impact of COVID-19 infections and vaccines on the reproductive system and menstruation. This study aimed to assess the impact of COVID-19 infection and vaccines on menstrual symptoms. METHODS: A cross-sectional survey utilizing face-to-face interviews from January 1 to 31 March 2022 was conducted in the city of Al-Karak in southern Jordan. The questionnaire included sociodemographic characteristics, medical and reproductive history, the contraceptive method used if any, menstrual cycle (MC) status, previous medical and drug history, and the impact of infection and vaccination on the MC. RESULTS: The study questionnaire was completed by 400 participants with a mean age of 32.1±12.6 years. Regarding the history of COVID-19 infections, 33.8% of the participants reported a history of confirmed COVID-19 infections, 77.8% of them did not report any menstrual changes following the infection, while the remaining 22.2% reported changes in menstruation. The most commonly reported post-COVID-19 manifestations were irregular (27.6%) and light menstrual cycle (MC) (24.15) or dysmenorrhea (24.1%). Heavy menstruation was reported by 17.2% of participants post-COVID-19 infection. Two-thirds of the study participants (66.6%) reported no changes in the MC following the administration of the COVID-19 vaccine. The most reported symptoms for those who experienced changes in the MC following the vaccination were irregular cycle (13.1%), heavy menstruation (7%), and light menstruation (7%). Other reported symptoms were dysmenorrhea (4.6%), intermenstrual bleeding (1.2%), and amenorrhea (0.5%). CONCLUSION: This study revealed minor changes in the MC following COVID-19 infections and administration of the COVID-19 vaccine. These findings are consistent with published reports. It is recommended that future clinical trials for new vaccines for women of childbearing age include outcomes related to sex hormones and MC. Women should be encouraged to take the vaccines and report symptoms to healthcare professionals for further assessment.

mRNA translation in astrocytes controls hippocampal long-term synaptic plasticity and memory.

Activation of neuronal protein synthesis upon learning is critical for the formation of long-term memory. Here, we report that learning in the contextual fear conditioning paradigm engenders a decrease in eIF2α (eukaryotic translation initiation factor 2) phosphorylation in astrocytes in the hippocampal CA1 region, which promotes protein synthesis. Genetic reduction of eIF2α phosphorylation in hippocampal astrocytes enhanced contextual and spatial memory and lowered the threshold for the induction of long-lasting plasticity by modulating synaptic transmission. Thus, learning-induced dephosphorylation of eIF2α in astrocytes bolsters hippocampal synaptic plasticity and consolidation of long-term memories.

COVID-19 pandemic impact on antibiotics sensitivity of E. coli and K. pneumoniae from urine specimens: a retrospective study.

INTRODUCTION: Escherichia coli and Klebsiella pneumoniae are common organisms associated with urinary tract infections. The COVID-19 pandemic has had a negative impact on antibiotics misuse globally. This study analyzed the antibiotic susceptibility for these two pathogens isolated from urine samples during the period of 18 months before and after the COVID-19 pandemic. METHODOLOGY: This retrospective study was conducted in Al-Karak government referral and teaching hospital in Jordan. The study included two groups; group A included urine samples from September 2018 to March 11, 2020, while group B from March 12, 2020 to August 2021. Samples were analyzed using the automated VITEK 2 system and the analysis of results was done using the WHONET version 5.6. RESULTS: A total of 642 E. coli and 113 K. pneumoniae were isolated and analyzed. The antibiogram showed a significant overall increase in antibiotic susceptibility of both bacteria during the pandemic period (group B). The sensitivity has significantly increased by 75% (15/20) and 50% (10/20) for all antibiotics used for E. coli and K. pneumoniae respectively. On the other hand, E. coli showed a significant increase in resistance to ceftriaxone (13.4%) and gentamicin (6.4%). A similar trend of an increase in resistance to gentamicin (17.4%) was also noticed among K. pneumoniae isolates. CONCLUSIONS: The antimicrobial susceptibility pattern for urine isolates showed an increased overall sensitivity and an increased resistance to ceftriaxone and gentamicin during the pandemic period. Our results highlight the need for revising and updating the antimicrobial stewardship programs post-COVID pandemic utilizing local data.

Immuno-affinity chromatography for purification of IgG from hyper-immune sera raised against 146S fraction of Foot and Mouth Disease Virus for diagnostic purposes.

Immunoaffinity chromatography (IAC) is a fundamental isolation and purification tool which is incorporated in a substantial range of therapeutic and diagnostic applications. This study has reappraised the usefulness of immunoaffinity chromatography for the purification of polyclonal antibodies. Protein A based IAC is a convenient and reliable method for purification of IgG, from hyperimmunesera (HIS) raised in experimental animals such as rabbits, guinea pigs and mice to be utilized in pharmaceutics and diagnostics. The 146S fraction of Foot and Mouth Disease virus (FMDV) TCID50=10 5.6 was cultured on a baby hamster kidney cell line 21 (BHK-21), concentrated using salt precipitation method using PEG 6000, purified by size exclusion chromatography (SEC) using Sepharose-30 at 254nm absorbance. Purification of 146S FMDV was analyzed using 12% SDS-PAGE which provided two bands of light and heavy chains. The alum-based vaccine, consisting of ≥10µg of 146S FMDV, was applied in 10 male rabbits and 10 male guinea pigs and two animals of each group were taken as a negative control. The titer of serum was calculated using virus neutralization test. A Protein-A kit (Thermo scientific- 44667, 0528.2) was used to purify HIS raised against 146S FMDV and validated using 12% SDS PAGE in reducing condition. The data demonstrate that protein-A affinity chromatography is an efficient tool for the purification of antibodies from hyper-immune sera raised against 146S FMDV and can be used for the production of diagnostic kits e.g. Enzyme linked immuno-sorbent assay (ELISA) and radioimmunoassay.

Willingness of Healthcare Workers to Recommend or Receive a Third COVID-19 Vaccine Dose: A Cross-Sectional Study from Jordan.

BACKGROUND: The availability of COVID-19 vaccines worldwide necessitates measuring healthcare workers' (HCWs') willingness to recommend or receive these vaccines. Therefore, we conducted a local study in Jordan to assess HCWs' willingness to recommend or receive a third dose of a COVID-19 vaccine and the predictors of such a decision. A cross-sectional study investigated Jordanian HCWs' willingness regarding a third dose of a COVID-19 vaccine using a self-administered online questionnaire through WhatsApp, a mobile phone application. A total of 300 HCWs participated in the current study. Of these HCWs, 65.3% were physicians, 25.3% were nurses, and 9.3% were pharmacists. HCWs' overall willingness regarding a third vaccine dose was 68.4% (49.4% certainly and 19.0% probably), whereas the overall willingness of HCWs to recommend a third dose to their patients was 73.3% (49.0% certainly and 24.3% probably). Males had significantly higher willingness than females (82.1% vs. 60.1%, p < 0.05). Physicians reported more willingness than nurses and pharmacists. HCWs' willingness was not significantly affected by direct contact with a patient infected with COVID-19 or by a personal history of COVID-19 infection. Only 31% of HCWs were certainly willing to recommend the vaccine to their patients with chronic diseases, and only 28% of the participants were certainly willing to recommend it to people aged 65 or older. HCWs' willingness to receive a third dose of a COVID-19 vaccine is limited in Jordan. This has affected their certainty in recommending this vaccine to their patients or people older than 60. Decision-makers and health-promotion programs in Jordan should focus on addressing this public health problem.

Deregulation of the cyclin-dependent kinase inhibitor p27 as a putative candidate for transformation in Chlamydia trachomatis infected mesenchymal stem cells.

Purpose: Several pathological conditions might cause the degradation of the cyclin-dependent kinase inhibitor (CKI) p27 and cell cycle arrest at the G1 phase, including cancers and infections. Chlamydia trachomatis (Ctr), as an obligatory intracellular pathogen, has been found to alter the fate of the cell from different aspects. In this study, we aimed to investigate the effect of Ctr infection on the expression of the important cell cycle regularity protein p27 in mesenchymal stem cells (MSCs). Methods: Isolation of MSCs from healthy human fallopian tube was confirmed by detection of the stemness markers Sox2, Nanog and Oct4 and the surface markers CD44, CD73 and CD90 by Western blotting and fluorescence-activated cell sorting analysis. The expression of p27 was downregulated at the protein level upon Ctr D infection measured by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR), IF and Western blotting. Recovery of p27 in Ctr D-infected MSCs was achieved by treatment with difluoromethylornithine (DFMO). Ctr D infected MSCs were able to produce colonies in anchorage-independent soft agar assay. Conclusion: Ctr D infection was able to downregulate the expression of the important cell cycle regulator protein p27, which will be considered a putative candidate for transformation in Ctr D infected MSCs.

The inhibitory NKR-P1B receptor regulates NK cell-mediated mammary tumor immunosurveillance in mice.

Natural killer (NK) cells are an important component of anti-cancer immunity, and their activity is regulated by an array of activating and inhibitory receptors. In mice, the inhibitory NKR-P1B receptor is expressed in NK cells and recognizes the C-type lectin-related protein-b (Clr-b) ligand. NKR-P1B:Clr-b interactions represent a 'missing-self' recognition system to monitor cellular levels of Clr-b on healthy and diseased cells. Here, we report an important role for NKR-P1B:Clr-b interactions in tumor immunosurveillance in MMTV-PyVT mice, which develop spontaneous mammary tumors. MMTV-PyVT mice on NKR-P1B-deficient genetic background developed mammary tumors earlier than on wild-type (WT) background. A greater proportion of tumor-infiltrating NK cells downregulate expression of the transcription factor Eomesodermin (EOMES) in NKR-P1B-deficient mice compared to WT mice. Tumor-infiltrating NK cells also downregulated CD49b expression but gain CD49a expression and exhibit effector functions, such as granzyme B upregulation and proliferation in mammary tumors. However, unlike the EOMES+ NK cells, the EOMES‒ NK cell subset is unable to respond to further in vitro stimulation and exhibits phenotypic alterations associated with immune dysfunction. These alterations included increased expression of PD-1, LAG-3, and TIGIT and decreased expression of NKp46, Ly49C/I, CD11b, and KLRG-1. Furthermore, tumor-infiltrating NKR-P1B-deficient NK cells exhibited an elevated dysfunctional immune phenotype compared to WT NK cells. These findings demonstrate that the NKR-P1B receptor plays an important role in mammary tumor surveillance by regulating anti-cancer immune responses and functional homeostasis in NK cells.

Genome analysis of novel Apilactobacillus sp. isolate from butterfly (Pieris canidia) gut reveals occurrence of unique glucanogenic traits and probiotic potential.

This study was conducted with a perception that fructose-rich niches may inhabit novel species of lactic acid bacteria that are gaining importance as probiotics and for the production of exopolysaccharides that have applications in food and pharmaceuticals. Recently, some Lactobacillus species have been reclassified as fructophilic lactic acid bacteria due to their preference for fructose over glucose as a carbon source. These bacteria are likely to be found in fructose rich niches such as flower nectar and insects that feed on it. We explored the butterfly gut and acquired a new isolate, designated as F1, of fructophilic lactic acid bacteria, which produces a glucan-type exopolysaccharide. Whole genome sequencing and in silico analysis revealed that F1 has significantly lower average nucleotide identity and DNA-DNA hybridization values as compared to its closest Apilactobacillus neighbors in phylogenetic analysis. Therefore, we declare the isolate F1 as a novel Apilactobacillus species with the proposed name of Apilactobacillus iqraium F1. Genome mining further revealed that F1 harbors genes for exopolysaccharide synthesis and health-promoting attributes. To this end, F1 is the only Apilactobacillus species harboring three diverse α-glucan-synthesis genes that cluster with different types of dextransucrases in the dendrogram. Moreover, many nutritional marker genes, as well as genes for epithelial cell adhesion and antimicrobial synthesis, were also detected suggesting the probiotic attributes of F1. Overall analysis suggests A. iqraium sp. F1 be a potential candidate for various health beneficial and pharmaceutical applications.

Evolutionary dynamics of topotype ME-SA/Ind-2001 of foot and mouth disease virus serotype-O in Pakistan: 2017-2022.

Background: During past few years, the Ind-2001 lineage of the Middle East-South Asia topotype (ME-SA) of the foot-and-mouth disease (FMD) virus has been implicated in FMD outbreaks in Pakistan. Aims: This work conducts a comprehensive evolutionary analysis of the Ind-2001 and Pan Asia II lineages, with a specific emphasis on their geographical distribution, lineage classification, and sub-lineage distribution within the region. Furthermore, it aims to expand our understanding of the conserved region of the VP1 protein. Methods: Total samples (n=50) were subjected to antigen detection ELISA and RT-PCR for serotype determination. Confirmed serotype-O isolates (n=17) underwent sequencing for lineage comparison, mutation impact assessment on the VP1 protein GH loop, 3D structure prediction, and further comparative analysis. Results: Isolates collected from 2017 to 2020 were identified as serotypes O/ME-SA/Pan Asia II ANT10 and O/ME-SA/Pak14. Notably, isolates collected from 2020 to 2022 belonged to a novel FMDV serotype O/ME-SA/Ind-2001e lineage. Phylogenetic analyses indicated that these strains were distinct from dominant contemporaneous strains which may challenge Pakistan's FMD control measures. These isolates exhibited variance in the VP1 epitope, specifically in amino acid residues 135-155, known to influence neutralizing antibody generation. Conclusion: Observed mutations suggest potential challenges to current vaccination efficacy against FMD. This emphasizes enhanced FMD surveillance and demonstrates that tracking the emergence of the O/ME-SA/Ind-2001e lineage is important for determining FMD control strategies in Asia.

Alveolar macrophage metabolic programming via a C-type lectin receptor protects against lipo-toxicity and cell death.

Alveolar macrophages (AM) hold lung homeostasis intact. In addition to the defense against inhaled pathogens and deleterious inflammation, AM also maintain pulmonary surfactant homeostasis, a vital lung function that prevents pulmonary alveolar proteinosis. Signals transmitted between AM and pneumocytes of the pulmonary niche coordinate these specialized functions. However, the mechanisms that guide the metabolic homeostasis of AM remain largely elusive. We show that the NK cell-associated receptor, NKR-P1B, is expressed by AM and is essential for metabolic programming. Nkrp1b-/- mice are vulnerable to pneumococcal infection due to an age-dependent collapse in the number of AM and the formation of lipid-laden AM. The AM of Nkrp1b-/- mice show increased uptake but defective metabolism of surfactant lipids. We identify a physical relay between AM and alveolar type-II pneumocytes that is dependent on pneumocyte Clr-g expression. These findings implicate the NKR-P1B:Clr-g signaling axis in AM-pneumocyte communication as being important for maintaining metabolism in AM.

A Systematic Review of Whether the Use of N95 Respirator Masks Decreases the Incidence of Cardiovascular Disease in the General Population.

The usage of masks such as the N95 has increased exponentially worldwide. With the ever-increasing global rates of cardiovascular disease, it is vital that preventative measures are adopted to help tackle this crisis. N95 masks have been promoted as health prevention odysseys in the battle against viruses such as COVID-19. A systematic review was conducted on whether the N95 masks could help improve our cardiovascular health. Our data sources included PubMed, Medline and Scopus. Eleven studies met the eligibility criteria to be included in the review. N95 mask usage led to increased reports of dyspnoea, however, no significant effect was seen on blood pressure. N95 masks also showed improvement in aortic parameters. While encouraging results were yielded, further focussed studies on the use of N95 masks and the effect on various cardiovascular parameters would help strengthen the association.

Evaluating the Suitability of 3D Bioprinted Samples for Experimental Radiotherapy: A Pilot Study.

Radiotherapy is an important component in the treatment of lung cancer, one of the most common cancers worldwide, frequently resulting in death within only a few years of diagnosis. In order to evaluate new therapeutic approaches and compare their efficiency with regard to tumour control at a pre-clinical stage, it is important to develop standardized samples which can serve as inter-institutional outcome controls, independent of differences in local technical parameters or specific techniques. Recent developments in 3D bioprinting techniques could provide a sophisticated solution to this challenge. We have conducted a pilot project to evaluate the suitability of standardized samples generated from 3D printed human lung cancer cells in radiotherapy studies. The samples were irradiated at high dose rates using both broad beam and microbeam techniques. We found the 3D printed constructs to be sufficiently mechanically stable for use in microbeam studies with peak doses up to 400 Gy to test for cytotoxicity, DNA damage, and cancer cell death in vitro. The results of this study show how 3D structures generated from human lung cancer cells in an additive printing process can be used to study the effects of radiotherapy in a standardized manner.

The Total Synthesis of Glycolipids from Streptococcus pneumoniae and a Re-evaluation of Their Immunological Activity.

Invariant natural killer (iNK) T cells, Type I iNKTs, are responsible for the production of pro-inflammatory cytokines which induce a systemic immune response. They are distinctive in possessing an semi-invariant T-cell receptor that recognizes glycolipid antigens presented by CD1d, a protein closely related to the class I major histocompatibility complex, conserved across multiple mammalian species in a class of proteins well-renowned for their high degree of polymorphism. This receptor's first potent identified antigen is the α-galactosylceramide, KRN7000, a synthetic glycosphingolipid closely related to those isolated from bacteria that were found on a Japanese marine sponge. A corresponding terrestrial antigen remained unidentified until two specific diacylglycerol-containing glycolipids, reported to activate iNKT cells, were isolated from Streptococcus pneumoniae. We report the total synthesis and immunological re-evaluation of these two glycolipids. The compounds are unable to meaningfully activate iNKT cells. Computational modelling shows that these ligands, while being capable of interacting with the CD1d receptor, create a different surface for the binary complex that makes formation of the ternary complex with the iNKT T-cell receptor difficult. Together these results suggest that the reported activity might have been due to an impurity in the original isolated sample and highlights the importance of taking care when reporting biological activity from isolated natural products.

Production of a high molecular weight levan by Bacillus paralicheniformis, an industrially and agriculturally important isolate from the buffalo grass rhizosphere.

A new exopolysaccharide (EPS) producing Gram-positive bacterium was isolated from the rhizosphere of Bouteloua dactyloides (buffalo grass) and its EPS product was structurally characterized. The isolate, designated as LB1-1A, was identified as Bacillus paralicheniformis based on 16S rRNA gene sequence and phylogenetic tree analysis. The EPS produced by LB1-1A was identified as a levan, having ß(2 → 6) linked backbone with ß(2 → 1) linkages at the branch points (4.66%). The isolate LB1-1A yielded large amount (~ 42 g/l) of levan having high weight average molecular weight (Mw) of 5.517 × 107 Da. The relatively low degree of branching and high molecular weight of this levan makes B. paralicheniformis LB1-1A a promising candidate for industrial applications.

Whole Genome Sequence Analysis of a Novel Apilactobacillus Species from Giant Honeybee (Apis dorsata) Gut Reveals Occurrence of Genetic Elements Coding Prebiotic and Probiotic Traits.

Apilactobacillus spp. are classified as obligate fructophilic lactic acid bacteria (FLAB) that inhabit fructose-rich niches such as honeybee gut. Lactic acid bacteria are an important component of the gut microbiome and play a crucial role in maintaining gut health. In this study, a new FLAB strain HBW1, capable of producing glucan-type exopolysaccharide, was isolated from giant honeybee (Apis dorsata) gut and subjected to whole genome sequencing (WHS) to determine its health-beneficial traits. The genome size of the isolate was 1.49 Mb with a GC content of 37.2%. For species level identity, 16S rDNA sequence similarity, genome to genome distance calculator (dDDH), and average nucleotide identity (ANI) values were calculated. Phylogenetic analysis showed that the isolate HBW1 belongs to the Apilactobacillus genus. The dDDH and ANI values in comparison with closely clustered Apilactobacillus kunkeei species were 52% and 93.10%, respectively. Based on these values, we concluded that HBW1 is a novel species of Apilactobacillus, and we propose the name Apilactobacillus waqarii HBW1 for it. Further, WHS data mining of HBW1 revealed that it harbors two glucosyltransferase genes for prebiotic glucan-type exopolysaccharide synthesis. Moreover, chaperon (clp) and methionine sulfoxide reductase (msrA, msrB, and msrC) genes as well as nutritional marker genes for folic acid (folD) and riboflavin biosynthesis (rib operon), important for conferring probiotic properties, were also detected. Occurrence of these genetic traits make HBW1 an excellent candidate for application to improve gut function.

Cancer vaccines: past, present and future; a review article.

Immunotherapy and vaccines have revolutionized disease treatment and prevention. Vaccines against infectious diseases have been in use for several decades. In contrast, only few cancer vaccines have been approved for human use. These include preventative vaccines against infectious agents associated with cancers, and therapeutic vaccines used as immunotherapy agents to treat cancers. Challenges in developing cancer vaccines include heterogeneity within and between cancer types, screening and identification of appropriate tumour-specific antigens, and the choice of vaccine delivery platforms. Recent advances in all of these areas and the lessons learnt from COVID-19 vaccines have significantly boosted interest in cancer vaccines. Further advances in these areas are expected to facilitate development of effective novel cancer vaccines. In this review, we aim to discuss the past, the present, and the future of cancer vaccines.

Loss of p53 Expression in Gastric Epithelial Cells of Helicobacter pylori-Infected Jordanian Patients.

Background: Around half of the global population is chronically infected with the stomach bacterium Helicobacter pylori, making it one of the most common chronic infections worldwide. H. pylori induces the production of reactive oxygen species, DNA damage, and accelerates the degradation of the tumor suppressor protein p53, which may lead to cancer development. In this study, we investigated the relationship between H. pylori infection and the expression of p53 in gastric mucosa in a group of patients from Jordan. Methods: In this retrospective case-control study, the epithelium of gastric glands in subjects chronically infected with H. pylori was examined for the expression of p53. Paraffin-embedded gastric biopsy samples from the archives for 50 Jordanian patients diagnosed with chronic H. pylori infection and 25 samples free of H. pylori infection and any other gastric abnormalities were selected. Samples were analyzed for the presence of H. pylori as well as p53 expression levels in the mucosa and submucosa by immunohistochemical analyses and Western blotting. Results: H. pylori was detected in the gastric tissues of infected individuals (n = 50); whereas, no H. pylori infection was detected in uninfected healthy individuals (n = 25) using immunohistochemistry. In contrast to the noninfected samples of gastric mucosa, no nuclear p53 expression was detected in the infected samples using immunohistochemistry. In addition, the levels of p53 in H. pylori-positive samples detected by Western blotting were significantly lower than those in the negative individuals. Conclusion: Our data reveal that p53 protein expression decreased in gastric mucosa of patients infected with H. pylori. The loss of this tumor suppressor may play a role in the increased risk for tumor initiation associated with H. pylori carriage.

Characterization of rotavirus strains isolated from children with acute gastroenteritis in Rawalpindi, Pakistan.

Diarrhea is the leading cause of childhood morbidity and mortality particularly in developing countries and rotavirus has been identified as the major pathogen associated with diarrheal infections. This study was conducted to detect genotypic distribution of predominant rotavirus strains circulating in children suffering from acute gastroenteritis in Rawalpindi, Pakistan. Stool specimens were collected from children ≤5 years of age, visiting Military Hospital, Rawalpindi, with signs and symptoms of acute gastroenteritis. Two hundred and eighty-four specimens were collected during the period from April 2017 to March 2018. Enzyme immunoassay was performed for detection of rotavirus and reverse transcription-PCR (RT-PCR) was carried out for amplification of VP7 and VP4 gene segments followed by multiplex PCR using genotype-specific primers. Out of 284 children, 71 were found rotavirus positive and among them, 54% were females and 46% males. Our findings showed 92% of infection among children ≤2 years of age, while, the peak age of rotavirus incidence was found to be 6-12 months. Although, rotavirus infection was observed throughout the year but frequency increased in winter. Subtype G1P[8] was more prevalent followed by G2P[4], G3P[8], and G4P[6] subtypes. The results of this study provide insight into the disease burden as well as information on rotavirus diversity which will be useful to develop future strategies to control and prevent diarrheal infections among children.

Clr-f expression regulates kidney immune and metabolic homeostasis.

The C-type lectin-related protein, Clr-f, encoded by Clec2h in the mouse NK gene complex (NKC), is a member of a family of immune regulatory lectins that guide immune responses at distinct tissues of the body. Clr-f is highly expressed in the kidney; however, its activity in this organ is unknown. To assess the requirement for Clr-f in kidney health and function, we generated a Clr-f-deficient mouse (Clr-f-/-) by targeted deletions in the Clec2h gene. Mice lacking Clr-f exhibited glomerular and tubular lesions, immunoglobulin and C3 complement protein renal deposits, and significant abdominal and ectopic lipid accumulation. Whole kidney transcriptional profile analysis of Clr-f-/- mice at 7, 13, and 24 weeks of age revealed a dynamic dysregulation in lipid metabolic processes, stress responses, and inflammatory mediators. Examination of the immune contribution to the pathologies of Clr-f-/- mouse kidneys identified elevated IL-12 and IFNγ in cells of the tubulointerstitium, and an infiltrating population of neutrophils and T and B lymphocytes. The presence of these insults in a Rag1-/-Clr-f-/- background reveals that Clr-f-/- mice are susceptible to a T and B lymphocyte-independent renal pathogenesis. Our data reveal a role for Clr-f in the maintenance of kidney immune and metabolic homeostasis.

Spontaneous Formation of 3D Breast Cancer Tissues on Electrospun Chitosan/Poly(ethylene oxide) Nanofibrous Scaffolds.

Three-dimensional (3D) tissue culture has attracted a great deal of attention as a result of the need to replace the conventional two-dimensional cell cultures with more meaningful methods, especially for understanding the sophisticated nature of native tumor microenvironments. However, most techniques for 3D tissue culture are laborious, expensive, and limited to spheroid formation. In this study, a low-cost and highly effective nanofibrous scaffold is presented for spontaneous formation of reproducible 3D breast cancer microtissues. Experimentally, aligned and non-aligned chitosan/poly(ethylene oxide) nanofibrous scaffolds were prepared at one of two chitosan concentrations (2 and 4 wt %) and various electrospinning parameters. The resulting fabricated scaffolds (C2P1 and C4P1) were structurally and morphologically characterized, as well as analyzed in silico. The obtained data suggest that the fiber diameter, surface roughness, and scaffold wettability are tunable and can be influenced based on the chitosan concentration, electrospinning conditions, and alignment mode. To test the usefulness of the fabricated scaffolds for 3D cell culture, a breast cancer cell line (MCF-7) was cultured on their surfaces and evaluated morphologically and biochemically. The obtained data showed a higher proliferation rate for cells grown on scaffolds compared to cells grown on two-dimensional adherent plates (tissue culture plate). The MTT assay revealed that the rate of cell proliferation on nanofibrous scaffolds is statistically significantly higher compared to tissue culture plate (P ≤ 0.001) after 14 days of culture. The formation of spheroids within the first few days of culture shows that the scaffolds effectively support 3D tissue culture from the outset of the experiment. Furthermore, 3D breast cancer tissues were spontaneously formed within 10 days of culture on aligned and non-aligned nanofibrous scaffolds, which suggests that the scaffolds imitate the in vivo extracellular matrix in the tumor microenvironment. Detailed mechanisms for the spontaneous formation of the 3D microtissues have been proposed. Our results suggest that scaffold surface topography significantly influences tissue formation and behavior of the cells.