Scanning Electron Microscopy.

Scanning electron microscopy (SEM) remains distinct in its ability to allow topographical visualization of structures. Key elements to consider for successful examination of biological specimens include appropriate preparative and imaging techniques. Chemical processing induces structural artifacts during specimen preparation, and several factors need to be considered when selecting fixation protocols to reduce these effects while retaining structures of interest. Particular care for proper dehydration of specimens is essential to minimize shrinkage and is necessary for placement under the high-vacuum environment required for routine operation of standard SEMs. Choice of substrate for mounting and coating specimens can reduce artifacts known as charging, and a basic understanding of microscope settings can optimize parameters to achieve desired results. This article describes fundamental techniques and tips for routine specimen preparation for a variety of biological specimens, preservation of labile or fragile structures, immune-labeling strategies, and microscope imaging parameters for optimal examination by SEM. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Chemical preparative techniques for preservation of biological specimens for examination by SEM Alternate Protocol 1: Practical considerations for the preparation of soft tissues Alternate Protocol 2: Removal of debris from the exoskeleton of invertebrates Alternate Protocol 3: Fixation of colonies grown on agar plates Alternate Protocol 4: Stabilization of polysaccharide structures with alcian blue and lysine Alternate Protocol 5: Preparation of non-adherent particulates in solution for SEM Support Protocol 1: Application of thin layer of adhesive on substrate to improve adherence Support Protocol 2: Poly-L-lysine coating specimen substrates for improved adherence Support Protocol 3: Microwave processing of biological specimens for examination by SEM Basic Protocol 2: Critical point drying of specimens Alternate Protocol 6: Chemical alternative to critical point drying Basic Protocol 3: Sputter coating Alternate Protocol 7: Improved bulk conductivity through "OTOTO" Basic Protocol 4: Immune-labeling strategies Alternate Protocol 8: Immune-labeling internal antigens with small gold probes Alternate protocol 9: Quantum dot or fluoronanogold preparations for correlative techniques Basic Protocol 5: Exposure of internal structures by mechanical fracturing Basic Protocol 6: Exposure of internal structures of tissues by fracturing with liquid nitrogen Basic Protocol 7: Anaglyph production from stereo pairs to produce 3D images.

Prevalence of carboxy-Δ8 -tetrahydrocannabiniol in antidoping samples.

Δ9 -Tetrahydrocannabinol (Δ9 -THC) is usually the primary psychoactive agent in cannabis preparations. Recently, products containing another isomer, Δ8 -tetrahydrocannabinol (Δ8 -THC), have become available for sale. Δ8 -THC exists naturally in the cannabis plant at very low concentrations; hence, the Δ8 -THC present in most of the above-mentioned products is likely to be manufactured synthetically. A surge in popularity of these products, coupled with little oversight to ensure purity and potency, has led to reports of adverse events. Workplace drug testing programs as well as many sporting organizations prohibit the use of cannabinoids. Carboxy-Δ9 -THC (Δ9 -THC-COOH) is the targeted urinary metabolite for detection of cannabis use. The proliferation of products containing Δ8 -THC, which metabolizes to Δ8 -THC-COOH, presents analytical complexity with respect to separation and quantification of the individual isomers as well as legal complexity with respect to lack of clarity around the legal status of Δ8 -THC. This study aims to estimate the prevalence of Δ8 -THC use in the athlete community by monitoring for Δ8 -THC-COOH in samples collected for antidoping. A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method was utilized to resolve Δ8 and Δ9 -THC-COOH. One thousand samples with a presumptive Δ9 -THC-COOH finding in routine screening were analyzed by the above LC-MS/MS method. Approximately 12% of samples contained Δ8 -THC-COOH at relative abundances between 5% and 100% of total carboxy-THC content.

Intraoperative Identification and Mosaicplasty in a Case of Femur Subchondral Osteoid Osteoma.

Osteoid osteomas (OOs) are non-malignant primary bone abnormalities marked by a central nidus surrounded by reactive sclerosis. They typically manifest as aggravated nocturnal pain that responds to non-steroidal anti-inflammatory drugs (NSAIDs). These growths are most frequently found within the intracortical bone and the diaphysis of elongated bones. Within the realm of uncommon conditions, intra-articular OOs (IAOOs) exhibit distinctive presentations, often leading to postponed or inaccurate diagnoses. We present a patient with OO at the distal femur, accessible through the knee joint, which was intraoperatively identified and localized using a needle pricking technique and treated by arthrotomy and mosaicplasty.

Leishmania genetic exchange is mediated by IgM natural antibodies.

Host factors that mediate Leishmania genetic exchange are not well defined. Here we demonstrate that natural IgM (IgMn)1-4 antibodies mediate parasite genetic exchange by inducing the transient formation of a spherical parasite clump that promotes parasite fusion and hybrid formation. We establish that IgMn from Leishmania-free animals binds to the surface of Leishmania parasites to induce significant changes in the expression of parasite transcripts and proteins. Leishmania binding to IgMn is partially lost after glycosidase treatment, although parasite surface phosphoglycans, including lipophosphoglycan, are not required for IgMn-induced parasite clumping. Notably, the transient formation of parasite clumps is essential for Leishmania hybridization in vitro. In vivo, we observed a 12-fold increase in hybrid formation in sand flies provided a second blood meal containing IgMn compared with controls. Furthermore, the generation of recombinant progeny from mating hybrids and parental lines were only observed in sand flies provided with IgMn. Both in vitro and in vivo IgM-induced Leishmania crosses resulted in full genome hybrids that show equal patterns of biparental contribution. Leishmania co-option of a host natural antibody to facilitate mating in the insect vector establishes a new paradigm of parasite-host-vector interdependence that contributes to parasite diversity and fitness by promoting genetic exchange.

A Case of Chronic Rupture of Achilles Tendon Managed Using a Combination of Multiple Surgical Techniques.

Achilles tendon rupture is a common injury that occurs due to sudden dorsiflexion of the plantar-flexed foot. Both acute and chronic ruptures are frequently misdiagnosed and mistreated. Acute Achilles tendon rupture commonly occurs in middle-aged individuals (30-40 years). Although several operative procedures are available for Achilles tendon repair, the management of choice remains controversial and debatable. A 27-year-old male came to our clinic complaining of pain over the left ankle for the last five months. History revealed trauma caused by a heavy metal object five months ago. Physical examination revealed tenderness and swelling over the left heel. Ankle plantar flexion was restricted, and painful and squeeze test was positive. Magnetic resonance imaging was suggestive of a tear of the Achilles tendon in the left ankle. Surgical management was done with multiple techniques which included flexor hallucis longus tendon graft augmentation, end-to-end suturing (Krackow technique), V-Y plasty, and bioabsorbable suture anchor. Although complications such as scar stiffness and wound gaping are common in such cases, the postoperative outcome was excellent in our case according to the American Orthopedic Foot and Ankle Score.

Impact of Biotin Supplementation on Human Chorionic Gonadotropin Immunoassays Utilizing Biotin-Streptavidin Binding Methods in Urine.

BACKGROUND: Human chorionic gonadotropin (hCG) detection is indicative of pregnancy and can be indicative of some forms of cancerous tumors. The hCG drug itself, however, is a performance enhancing substance used by male athletes to increase testosterone production. Antidoping testing for hCG is conducted in urine, often on immunoanalyzer platforms, many of which utilize biotin-streptavidin dependent immunoassays in which the presence of biotin in samples is a known confounding factor. While biotin interference in serum has been well-studied, the extent of biotin interference in urine has not. METHODS: Ten active male individuals underwent a 2-week hCG administration protocol concurrent with supplementation with biotin (20 mg/day) or placebo. Urine and serum samples were collected throughout the study and analyzed for hCG and biotin concentrations. RESULTS: Urinary biotin levels in the hCG + biotin group increased 500-fold over baseline and 29-fold over corresponding serum biotin levels after biotin supplementation. When using a biotin-dependent immunoassay, the hCG + placebo group produced hCG-positive results (hCG ≥ 5 mIU/mL) in 71% of urine samples, while the hCG + biotin group produced positive results in only 19% of samples. Both groups had elevated hCG values in serum measurements by a biotin-dependent immunoassay and in urine when using a biotin-independent immunoassay. Urinary hCG measurements and biotin levels from the hCG + biotin group showed a negative correlation (Spearman r = -0.46, P < 0.0001) when measured using a biotin-dependent immunoassay. CONCLUSIONS: Biotin supplementation can severely suppress urinary hCG values in assays utilizing biotin-streptavidin binding methods and therefore these types of assays are not recommended for use in urine samples containing high levels of biotin. Clinicaltrials.gov Registration Number: NCT05450900.

δ13 C values of urinary 19-norandrosterone in antidoping samples and potential for adverse findings from boar offal consumption.

19-Norandrosterone (19NA) is the preferred urinary target compound to identify doping with nandrolone or related 19-norsteroids. At concentrations between 2.5 and 15 ng/mL, isotope ratio mass spectrometry (IRMS) is required to establish exogenous origin of urinary 19NA. An absolute difference of 3‰ between urinary 19NA and an endogenous reference compound (ERC) constitutes a finding for exogenous origin of 19NA. Over the last 3 years, 77 samples containing urinary 19NA between 2.5 and 15 ng/mL were analyzed at our laboratory. The measured δ13 C values for 19NA ranged from -29.5‰ to -16.8‰. In comparison, the δ13 C values for the corresponding urinary ERCs ranged from -22.4‰ to -16.2‰. Due to the considerable overlap in values between the target compound and the natural range of urinary ERCs, it can be challenging to distinguish between endogenous and exogenous origins of urinary 19NA. In addition, it is well known that consumption of offal from non-castrated pigs can produce 19NA in urine. To determine whether this could cause a positive IRMS finding under the current IRMS positivity criteria, meat from non-castrated boars fed a mixture of corn and soy was consumed by 13 volunteers. Two volunteers produced 19NA findings above 2.5 ng/mL, and the measured isotope values, while inconsistent with documented 19-norsteroid preparations, did meet IRMS positivity criteria. However, these increases in 19NA urinary concentrations were short-lived due to rapid elimination. Timely follow-up collections may help support a claim for dietary exposure when low urinary concentrations of 19NA with pseudo-endogenous isotope values are observed.

Assessment of urban air quality from Twitter communication using self-attention network and a multilayer classification model.

Social media platforms are one of the prominent new-age methods used by public for spreading awareness or drawing attention on an issue or concern. This study demonstrates how the twitter responses of public can be used for qualitative monitoring of air pollution in an urban area. Tweets discussing about air quality in Delhi, India, were extracted during 2019-2020 using a machine learning technique based on self-attention network. These tweets were cleaned, sorted, and classified into 3-class quality viz. poor air quality, good air quality, and noise or neutral tweets. The present study used a multilayer classification model with first layer as an embedding layer and second layer as bi-directional long-short term memory (BiLSTM) layer. A method was then devised for estimating PM2.5 concentration from the tweets using 'spaCy' similarity analysis of classified tweets and data extracted from Continuous Ambient Air Quality Monitoring Stations (CAAQMS) in Delhi for the study period. The accuracy of this estimation was found to be high (80-99%) for extreme air quality conditions (extremely good or severe) and lower during moderate variations in air quality. Application of this methodology depended on perceivable changes in air quality, twitter engagement, and environmental consciousness among public.

Using the Sleeping Beauty (SB) Transposon to Generate Stable Cells Producing Enveloped Virus-Like Particles (eVLPs) Pseudotyped with SARS-CoV-2 Proteins for Vaccination.

The Sleeping Beauty (SB) transposon system is an efficient non-viral tool for gene transfer into a variety of cells, including human cells. Through a cut-and-paste mechanism, your favorite gene (YFG) is integrated into AT-rich regions within the genome, providing stable long-term expression of the transfected gene. The SB system is evolving and has become a powerful tool for gene therapy. There are no safety concerns using this system, the handling is easy, and the time required to obtain a stable cell line is significantly reduced compared to other systems currently available. Here, we present a novel application of this system to generate, within 8 days, a stable producer HEK293T cell line capable of constitutively delivering enveloped virus-like particles (eVLPs) for vaccination. We provide step-by-step protocols for generation of the SB transposon constructs, transfection procedures, and validation of the produced eVLPs. We next describe a method to pseudotype the constitutively produced eVLPs using the Spike protein derived from the SARS-CoV-2 virus (by coating the eVLP capsid with the heterologous antigen). We also describe optimization methods to scale up the production of pseudotyped eVLPs in a laboratory setting (from 100 µg to 5 mg). © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Generation of the SB plasmids Basic Protocol 2: Generation of a stable HEK293T cell line constitutively secreting MLV-based eVLPs Basic Protocol 3: Evaluation of the SB constructs by immunofluorescence assay Basic Protocol 4: Validation of eVLPs by denaturing PAGE and western blot Alternate Protocol 1: Analysis of SARS-CoV-2 Spike protein oligomerization using blue native gel electrophoresis and western blot Alternate Protocol 2: Evaluation of eVLP quality by electron microscopy (negative staining) Basic Protocol 5: Small-scale production of eVLPs Alternate Protocol 3: Large-scale production of eVLPs (up to about 1 to 3 mg VLPs) Alternate Protocol 4: Large-scale production of eVLPs (up to about 3 to 5 mg VLPs) Support Protocol: Quantification of total protein concentration by Bradford assay.

Correlation between placental histopathology and perinatal outcome in COVID-19.

Objectives: An alarming rate of adverse perinatal outcomes as well as maternal deaths has been reported worldwide during this pandemic. It would be prudent to start thinking on the lines of acute or chronic intrauterine fetal hypoxia due to placental microvascular pathology or villitis caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection. Autopsy studies of deceased patients with severe COVID-19 have revealed the presence of diffuse pulmonary alveolar damage, thrombosis, and microvascular injuries. It is expected that similar pathological features such as microvascular injuries could be found in the placenta of infected pregnant women. Materials and Methods: Placentas of singleton pregnancies from 42 SARS-CoV-2 positive mothers delivered at term were submitted for histopathological examination. Those with multifetal gestation, hypertensive disorder, fetal growth restriction, structural or chromosomal anomalies in the fetus, thrombophilia, prolonged prelabor rupture of membranes, and placenta accreta spectrum were excluded from the study. Histopathological examination was done by two pathologists independently and only those results concurred by both were reported. Histopathological features and corresponding neonatal outcome were analyzed. Results: Reports of 42 placentas from patients with SARS-CoV-2, delivered at term (37-40 weeks) were analyzed in our study. Features of maternal vascular malperfusions (MVM) were present in 45% (n = 19) cases. Features of fetal vascular malperfusions (FVM) were present in 23.8% (n = 10) cases. There were 47.6% (n = 20) cases showing at least one feature of acute inflammatory pathology (AIP) and 42.8% (n = 18) showing features of chronic inflammatory pathology (CIP). Neonatal respiratory distress syndrome was found in 19% (n = 8) of the neonates. Correspondingly, nearly all placentas (n = 7) of these neonates showed features of MVM, FVM, AIP and CIP. There was no maternal or neonatal mortality in our study group. Conclusion: The main findings of our study include maternal as well as fetal vascular malperfusions and placental inflammatory pathology. These findings provide an outline for better understanding of etiological factors and pathogenesis of adverse perinatal outcomes in SARS-CoV-2 infection.

Loss of GdpP Function in Staphylococcus aureus Leads to ß-Lactam Tolerance and Enhanced Evolution of ß-Lactam Resistance.

Infections caused by Staphylococcus aureus are a leading cause of mortality. Treating infections caused by S. aureus is difficult due to resistance against most traditional antibiotics, including ß-lactams. We previously reported the presence of mutations in gdpP among S. aureus strains that were obtained by serial passaging in ß-lactam drugs. Similar mutations have recently been reported in natural S. aureus isolates that are either nonsusceptible or resistant to ß-lactam antibiotics. gdpP codes for a phosphodiesterase that cleaves cyclic-di-AMP (CDA), a newly discovered second messenger. In this study, we sought to identify the role of gdpP in ß-lactam resistance in S. aureus. Our results showed that gdpP-associated mutations caused loss of phosphodiesterase function, leading to increased CDA accumulation in the bacterial cytosol. Deletion of gdpP led to an enhanced ability of the bacteria to withstand a ß-lactam challenge (2 to 3 log increase in bacterial CFU) by promoting tolerance without enhancing MICs of ß-lactam antibiotics. Our results demonstrated that increased drug tolerance due to loss of GdpP function can provide a selective advantage in acquisition of high-level ß-lactam resistance. Loss of GdpP function thus increases tolerance to ß-lactams that can lead to its therapy failure and can permit ß-lactam resistance to occur more readily.

Detection of testosterone microdosing in healthy females.

The ready detectability of synthetic androgens by mass spectrometry (MS)-based antidoping tests has reoriented androgen doping to using testosterone (T), which must be distinguished from its endogenous counterpart making detection of exogenous T harder. We investigated urine and serum steroid and hematological profiling individually and combined to determine the optimal detection model for T administration in women. Twelve healthy females provided six paired blood and urine samples over 2 weeks prior to treatment consisting of 12.5-mg T in a topical transdermal gel applied daily for 7 days. Paired blood and urine samples were then obtained at the end of treatment and Days 1, 2, 4, 7, and 14 days later. Compliance with treatment and sampling was high, and no adverse effects were reported. T treatment significantly increased serum and urine T, serum dihydrotestosterone (DHT), urine 5α-androstane-3α,17ß-diol (5α-diol) epitestosterone (E), and urine T/E ratio with a brief window of detection (2-4 days) as well as total and immature (medium and high fluorescence) reticulocytes that remained elevated over the full 14 posttreatment days. Carbon isotope ratio MS and the OFF score and Abnormal Blood Profile score (ABPS) were not discriminatory. The optimal multivariate model to identify T exposure combined serum T, urine T/E ratio with three hematological variables (% high fluorescence reticulocytes, mean corpuscular hemoglobin, and volume) with the five variables providing 93% correct classification (4% false positive, 10% false negatives). Hence, combining select serum and urine steroid MS variables with reticulocyte measures can achieve a high but imperfect detection of T administration to healthy females.

Minimally Invasive Coracoclavicular Ligament Reconstruction Using Semitendinosus Autograft in a Case of Cho's Type IIC Lateral End Clavicle Fracture With Torn Conoid and Trapezoid Ligaments.

Clavicle fractures are a common clinical problem that accounts for about 10% of all fractures. Cho's type II fractures compromise the integrity of the coracoclavicular ligament and are thus inherently unstable, necessitating a lengthy healing period and being associated with a high rate of nonunion or malunion. The lowering of these rates is largely dependent on restoring the stability of the distal clavicle. In our case report, a 60-year-old male came to the OPD with complaints of pain over the right shoulder for two days following a fall with his arm in an adducted position. He also complained of an inability to abduct the right shoulder beyond 45 degrees. A plain radiograph was done, which was suggestive of a right lateral end clavicle fracture with increased coracoclavicular distance compared to the uninvolved shoulder. He was taken up for surgery after routine laboratory investigations and pre-anaesthesia check-up and minimally invasive coracoclavicular ligament reconstruction was done using a semitendinosus autograft. Intra-operatively, both the conoid and trapezoid ligaments showed tears. A universal shoulder immobilizer was applied post-surgery and continued for six weeks. Pendular exercises of the shoulder were started as per tolerance, and the patient responded well to surgery.

Ovarian Ligament Plication as a Treatment for Patient with Elongated Ovarian Ligament with Recurrent Abdominal Pain in the Absence of Ovarian Torsion.

Elongated ovarian ligament can lead to adnexal torsion. Several cases of ovarian torsion have been reported where the ovarian ligament was elongated, and ovarian detorsion and ovarian ligament plication were done. In our case, a young girl presented with recurrent left lower abdominal pain, especially after exercise, with normal ovaries found on ultrasound. Laparoscopy was performed given recurrent pelvic pain and found elongated left ovarian ligament with normal ovaries. Considering the possibility of recurrent torsion and detorsion of the ovary due to elongated left ovarian ligament, left ovarian ligament plication was done. The patient remained pain-free till 1 year of follow-up. No such case has been reported in the literature where the ovarian ligament plication was performed without torsion. Hence, elongated ovarian ligament could cause recurrent pelvic pain due to possible torsion, and a simple, easy procedure of ovarian ligament plication can help relieve pain.

A Rare Case of Destruction of the First Metatarsophalangeal Joint in a Patient With Gout.

Gout is a crystal deposition disorder caused due to the deposition of monosodium urate crystals in joints and other tissues secondary to hyperuricemia. Podagra is the term for gout of the first metatarsophalangeal joint. In our case report, a 30-year-old male patient came to our OPD with complaints of swelling over the first metatarsophalangeal joint for one year, which was insidious in onset, localized, and had a sudden increase in size over the past three months. The patient also complained of an inability to properly wear his shoe. A plain radiograph was done, which was suggestive of an expansile lesion with the destruction of the first metatarsophalangeal joint and the erosion of the joint surface extending to the head of the first metatarsal and the proximal phalanx of the great toe. Lab investigations revealed a serum uric acid level of 10.2 mg/dl and an acid phosphatase level of 8.92 U/L. Excision of the lesion was done and a frozen section biopsy was sent intra-operatively which confirmed the presence of monosodium urate crystals. A fibular strut graft was taken to fill the defect using a square nail passing through the first metatarsophalangeal joint and a Kirschner wire was added to the interphalangeal joint to maintain the stability of the reduction. The foot was immobilized for six weeks following which the Kirschner wire was removed and range-of-motion exercises started. There was no residual deformity, and the patient responded well to the treatment.

Rottlerin inhibits La Crosse virus-induced encephalitis in mice and blocks release of replicating virus from the Golgi body in neurons.

La Crosse virus (LACV) is a mosquito-borne orthobunyavirus that causes approximately 60 to 80 hospitalized pediatric encephalitis cases in the United States yearly. The primary treatment for most viral encephalitis, including LACV, is palliative care, and specific antiviral therapeutics are needed. We screened the National Center for Advancing Translational Sciences library of 3,833 FDA-approved and bioactive small molecules for the ability to inhibit LACV-induced death in SH-SY5Y neuronal cells. The top three hits from the initial screen were validated by examining their ability to inhibit virus-induced cell death in multiple neuronal cell lines. Rottlerin consistently reduced LACV-induced death by 50% in multiple human and mouse neuronal cell lines with an effective concentration of 0.16-0.69 µg ml-1 depending on cell line. Rottlerin was effective up to 12 hours post-infection in vitro and inhibited virus particle trafficking from the Golgi apparatus to trans-Golgi vesicles. In human inducible pluripotent stem cell-derived cerebral organoids, rottlerin reduced virus production by one log and cell death by 35% compared with dimethyl sulfoxide-treated controls. Administration of rottlerin in mice by intraperitoneal or intracranial routes starting at 3 days post-infection decreased disease development by 30-50%. Furthermore, rottlerin also inhibited virus replication of other pathogenic California serogroup orthobunyaviruses (Jamestown Canyon and Tahyna virus) in neuronal cell lines.

Extraperitoneal Laparoscopy in Severe Intra-abdominal Adhesions: A Safe Alternative to Laparotomy.

Surgery in a frozen abdomen can be difficult and dangerous with a significant risk of visceral injuries. We report a case of a 26-year-old lady with chronic pelvic pain diagnosed to have large bilateral adnexal cysts on magnetic resonance imaging with normal tumor markers. She had previous two laparotomies for benign conditions. Laparoscopy was planned, but pneumoperitoneum could not be created due to dense intraperitoneal adhesions. Direct entry was done into the preperitoneal space followed by insufflation of gas in this space. Blunt and sharp dissection of this space was done without breaching the peritoneum to reach the adnexa. The adnexal cyst was found to be encysted collection due to adhesions from previous surgeries. Deroofing was done followed by the visualization of pelvic structures intraperitoneally. Extraperitoneal laparoscopy may be used as a safe alternative to laparotomy in patients with dense intra-abdominal adhesions with the advantage of faster postoperative recovery.

Age influences susceptibility of brain capillary endothelial cells to La Crosse virus infection and cell death.

BACKGROUND: A key factor in the development of viral encephalitis is a virus crossing the blood-brain barrier (BBB). We have previously shown that age-related susceptibility of mice to the La Crosse virus (LACV), the leading cause of pediatric arbovirus encephalitis in the USA, was associated with the ability of the virus to cross the BBB. LACV infection in weanling mice (aged around 3 weeks) results in vascular leakage in the olfactory bulb/tract (OB/OT) region of the brain, which is not observed in adult mice aged > 6-8 weeks. Thus, we studied age-specific differences in the response of brain capillary endothelial cells (BCECs) to LACV infection. METHODS: To examine mechanisms of LACV-induced BBB breakdown and infection of the CNS, we analyzed BCECs directly isolated from weanling and adult mice as well as established a model where these cells were infected in vitro and cultured for a short period to determine susceptibility to virus infection and cell death. Additionally, we utilized correlative light electron microscopy (CLEM) to examine whether changes in cell morphology and function were also observed in BCECs in vivo. RESULTS: BCECs from weanling, but not adult mice, had detectable infection after several days in culture when taken ex vivo from infected mice suggesting that these cells could be infected in vitro. Further analysis of BCECs from uninfected mice, infected in vitro, showed that weanling BCECs were more susceptible to virus infection than adult BCECs, with higher levels of infected cells, released virus as well as cytopathic effects (CPE) and cell death. Although direct LACV infection is not detected in the weanling BCECs, CLEM analysis of brain tissue from weanling mice indicated that LACV infection induced significant cerebrovascular damage which allowed virus-sized particles to enter the brain parenchyma. CONCLUSIONS: These findings indicate that BCECs isolated from adult and weanling mice have differential viral load, infectivity, and susceptibility to LACV. These age-related differences in susceptibility may strongly influence LACV-induced BBB leakage and neurovascular damage allowing virus invasion of the CNS and the development of neurological disease.

Cytosolic replication in epithelial cells fuels intestinal expansion and chronic fecal shedding of Salmonella Typhimurium.

Persistent and intermittent fecal shedding, hallmarks of Salmonella infections, are important for fecal-oral transmission. In the intestine, Salmonella enterica serovar Typhimurium (STm) actively invades intestinal epithelial cells (IECs) and survives in the Salmonella-containing vacuole (SCV) and the cell cytosol. Cytosolic STm replicate rapidly, express invasion factors, and induce extrusion of infected epithelial cells into the intestinal lumen. Here, we engineered STm that self-destruct in the cytosol (STmCytoKill), but replicates normally in the SCV, to examine the role of cytosolic STm in infection. Intestinal expansion and fecal shedding of STmCytoKill are impaired in mouse models of infection. We propose a model whereby repeated rounds of invasion, cytosolic replication, and release of invasive STm from extruded IECs fuels the high luminal density required for fecal shedding.

Feasibility and efficacy of modern minilaparoscopy with 2.9 mm laparoscope for diagnostic and level II gynaecological procedure.

OBJECTIVE: Laparoscopy has now become a state-of-the-art technique for many diagnostic and therapeutic procedures with known advantages over laparotomy. There is scarce literature from India regarding minilaparoscopy, as per our literature review. Therefore, we performed this study with a 2.9-mm laparoscope to determine its feasibility and efficacy for diagnostic purposes and level II surgeries with the aim of reducing postoperative pain and better cosmesis. METHODS: This was a prospective study conducted from June 2019 to March 2020. Diagnostic modern minilaparoscopy with a 2.9-mm telescope was performed under general anesthesia by a single surgeon. Operative intervention was performed depending on the intraoperative findings. RESULTS: The mean age was 29.3 years. The most common indication for laparoscopy was infertility (98%). Only diagnostic laparoscopy was performed in 76% of patients, while 24% underwent operative laparoscopy. The various operative procedures performed were cystectomy, salpingectomy, ovarian drilling, and adhesiolysis. The mean visual analog scale scores at 1 hour and 2 hours postoperatively and discharge were 1.57±0.59, 1.41±0.51, and 1.29±0.47, respectively. Mild pain was present in 70 (72.2%) patients at the time of discharge, and only one patient had severe pain. Five or more analgesic tablets were required in only 16.5% of patients in the postoperative period. There was no wound infection or port-site hernia at follow-up. CONCLUSION: Modern minilaparoscopy with a 2.9-mm laparoscope is a feasible and safe option for diagnostic laparoscopy and level II gynecological procedures with minimal postoperative morbidity, such as pain and wound infection, and provides good cosmetic outcomes.