Genetic modification of spermatogonial stem cells (SSCs) is an alternative method to pronuclear microinjection and somatic cell nuclear transfer for transgenesis in large animals. In the present ...study, we optimized the process of homologous SSC transplantation in the water buffalo (Bubalus bubalis) using transfected enriched SSCs generated by a non-viral transfection approach. Firstly, the SSC enrichment efficiencies of extracellular matrix components viz. collagen, gelatin, and Datura stramonium agglutinin (DSA) lectin were determined either individually or in combination with Percoll density gradient centrifugation. The highest enrichment was achieved after differential plating with DSA lectin followed by Percoll density gradient centrifugation. Nucleofection showed greater transfection efficiency (68.55 ± 4.56%, P < 0.05) for enriched SSCs in comparison to fugene HD (6.7 ± 0.25%) and lipofectamine 3000 (15.57 ± 0.74%). The transfected enriched SSCs were transplanted into buffalo males under the ultrasound guidance and testis was removed by castration after 7-8 weeks of transplantation. Persistence and localization of donor cells within recipient seminiferous tubules was confirmed using fluorescent microscopy. Further confirmation was done by flow cytometric evaluation of GFP expressing cells among those isolated from two-step enzymatic digestion of recipient testicular parenchyma. In conclusion, we demonstrated for the first time, generation of buffalo transfected enriched SSCs and their successful homologous transplantation in buffaloes. This study represents the first step towards genetic modifications in buffaloes using SSC transplantation technique.
•For the first time, homologous SSC transplantation was attempted in the water buffalo.•An efficient SSC enrichment protocol was developed by comparing the efficiencies of various enrichment procedures.•The combined use of DSA lectin and Percoll density gradient centrifugation resulted in the highest SSC enrichment.•Nucleofection offered better transfection efficiency than lipofection for buffalo enriched SSCs.•Transfected enriched SSCs following homologous transplantation could successfully colonize the recipient testes.
The cytolytic activity of NK cells is tightly regulated by inhibitory receptors specific for MHC class I Ags. We have investigated the composition of signal transduction molecules in the ...supramolecular activation clusters in the MHC class I-regulated cytolytic and noncytolytic NK cell immune synapses. KIR2DL3-positive NK clones that are specifically inhibited in their cytotoxicity by HLA-Cw*0304 and polyclonal human NK cells were used for conjugate formation with target cells that are either protected or are susceptible to NK cell-mediated cytotoxicity. Polarization of talin, microtubule-organizing center, and lysosomes occurred only during cytolytic interactions. The NK immune synapses were analyzed by three-dimensional immunofluorescence microscopy, which showed two distinctly different synaptic organizations in NK cells during cytolytic and noncytolytic interactions. The center of a cytolytic synapse with MHC class I-deficient target is comprised of a complex of signaling molecules including Src homology (SH)2-containing protein tyrosine phosphatase-1 (SHP-1). Closely related molecules with overlapping functions, such as the Syk kinases, SYK, and ZAP-70, and adaptor molecules, SH2 domain-containing leukocyte protein of 76 kDa and B cell linker protein, are expressed in activated NK cells and are all recruited to the center of the cytolytic synapse. In contrast, the noncytolytic synapse contains SHP-1, but is lacking other components of the central supramolecular activation cluster. These findings indicate a functional role for SHP-1 in both the cytolytic and noncytolytic interactions. We also demonstrate, in three-cell conjugates, that a single NK cell forms a cytolytic synapse with a susceptible target cell in the presence of both susceptible and nonsusceptible target cells.
For inoperable stage I (T1-T2N0) small cell lung cancer (SCLC), national guidelines recommend chemotherapy with or without conventionally fractionated radiation therapy. The present ...multi-institutional cohort study investigated the role of stereotactic ablative radiation therapy (SABR) for this population.
The clinical and treatment characteristics, toxicities, outcomes, and patterns of failure were assessed in patients with histologically confirmed stage T1-T2N0M0 SCLC. Kaplan-Meier analysis was used to evaluate the survival outcomes. Univariate and multivariate analyses identified predictors of outcomes.
From 24 institutions, 76 lesions were treated in 74 patients (median follow-up 18 months). The median age and tumor size was 72 years and 2.5 cm, respectively. Chemotherapy and prophylactic cranial irradiation were delivered in 56% and 23% of cases, respectively. The median SABR dose and fractionation was 50 Gy and 5 fractions. The 1- and 3-year local control rate was 97.4% and 96.1%, respectively. The median disease-free survival (DFS) duration was 49.7 months. The DFS rate was 58.3% and 53.2% at 1 and 3 years, respectively. The median, 1-year, and 3-year disease-specific survival was 52.3 months, 84.5%, and 64.4%, respectively. The median, 1-year, and 3-year overall survival (OS) was 17.8 months, 69.9%, and 34.0% respectively. Patients receiving chemotherapy experienced an increased median DFS (61.3 vs 9.0 months; P=.02) and OS (31.4 vs 14.3 months; P=.02). The receipt of chemotherapy independently predicted better outcomes for DFS/OS on multivariate analysis (P=.01). Toxicities were uncommon; 5.2% experienced grade ≥2 pneumonitis. Post-treatment failure was most commonly distant (45.8% of recurrence), followed by nodal (25.0%) and "elsewhere lung" (20.8%). The median time to each was 5 to 7 months.
From the findings of the largest report of SABR for stage T1-T2N0 SCLC to date, SABR (≥50 Gy) with chemotherapy should be considered a standard option.
Somatic cell nuclear transfer (SCNT), using transgenic donor cells, is a highly efficient method for producing transgenic embryos. We compared the developmental competence, quality and gene ...expression of transgenic embryos produced by Hand-made cloning from buffalo fetal fibroblasts (BFFs) containing human insulin gene, with non-transgenic embryos produced from BFFs (Controls). The expression vector (pAcISUBC), constructed by inserting human insulin gene between DNA fragments containing mammary gland-specific buffalo β-lactoglobulin (buBLG) promoter and terminator buBLG 3′UTR regions into pAcGFP-N1 vector, was used for obtaining the 11 kb insert for transfection of BFFs by nucleofection. Presence of the transgene in embryos was confirmed by examining GFP expression by RT-PCR and immunofluorescence. The blastocyst rate was lower (P < 0.05) for transgenic embryos than for controls (35.7 ± 1.8% vs 48.7 ± 2.4%). The apoptotic index was higher (P < 0.05) for transgenic than for control blastocysts which, in turn, was higher (P < 0.05) than for IVF counterparts (6.9 ± 0.9, 3.8 ± 0.5 and 1.8 ± 0.3, respectively). The total cell number was similar for transgenic and non-transgenic blastocysts (143.2 ± 17.0 and 137.2 ± 7.6, respectively). The expression level of pro-apoptotic genes BAX and BID but not that of CASP3 and CASP9, and cell cycle check point control-related gene P53 was higher (P < 0.05), and that of development- (IGF-1R and G6PD) and pluripotency-related gene NANOG was lower (P < 0.05) in transgenic than in control embryos. The expression level of epigenetic-related genes DNMT1, DNMT3a and HDAC1 and pluripotency-related gene OCT4 was similar in the two groups. The expression level of BAX, BID, CASP9, P53, DNMT1 and DNMT3a was higher (P < 0.05) and that of OCT4, NANOG IGF-1R and G6PD was lower (P < 0.05) in cloned transgenic than in IVF blastocysts whereas, that of CASP3 and HDAC1 was similar between the two groups. In conclusion, these results suggest that transgenic embryos produced by SCNT have lower developmental competence and quality, and altered gene expression compared to non-transgenic embryos.
•Transgenic embryos having human insulin gene were produced by SCNT.•Transgenic embryos have lower blastocyst rate than non-transgenic controls.•Transgenic blastocysts have higher level of apoptosis than controls.•Gene expression is altered in transgenic compared to non-transgenic embryos.
The eLife Early-Career Advisory Group discusses eLife's new peer review and publishing model, and how the whole process of scientific communication could be improved for the benefit of early-career ...researchers and the entire scientific community.
TGFBI-related corneal dystrophy (CD) is characterized by the accumulation of insoluble protein deposits in the corneal tissues, eventually leading to progressive corneal opacity. Here we show that ...ATP-independent amyloid-β chaperone L-PGDS can effectively disaggregate corneal amyloids in surgically excised human cornea of TGFBI-CD patients and release trapped amyloid hallmark proteins. Since the mechanism of amyloid disassembly by ATP-independent chaperones is unknown, we reconstructed atomic models of the amyloids self-assembled from TGFBIp-derived peptides and their complex with L-PGDS using cryo-EM and NMR. We show that L-PGDS specifically recognizes structurally frustrated regions in the amyloids and releases those frustrations. The released free energy increases the chaperone's binding affinity to amyloids, resulting in local restructuring and breakage of amyloids to protofibrils. Our mechanistic model provides insights into the alternative source of energy utilized by ATP-independent disaggregases and highlights the possibility of using these chaperones as treatment strategies for different types of amyloid-related diseases.
Objective. Little is known about diabetes status among US blacks by nativity. This study aims to measure differences in diabetes among US blacks by region of birth and examines potential explanations ...for subgroup differences.
Design. Data from 47,751 blacks aged 25-74 pooled from the 2000-2013 waves of the National Health Interview Survey were analyzed. Logistic regression models predicted self-reported diabetes. The roles of education, income, body mass index (BMI), smoking, and duration of US residence were explored.
Results. Compared to the US-born, foreign-born blacks had significantly lower reported diabetes prevalence (8.94% vs. 11.84%) and diabetes odds ratio OR: 0.75; 95% confidence interval (CI): 0.62, 0.89, adjusting for socio-demographic characteristics. Further inclusion of education, income, household size, and smoking did not appreciably change the OR (0.77; 95% CI: 0.61, 0.86). Including an adjustment for BMI entirely eliminated the foreign-born advantage (OR 0.93; 95% CI: 0.78, 1.11). The foreign-born from the Caribbean/Americas had similar diabetes odds compared to the African-born. Among the foreign-born, an increased duration of US residence was associated with a higher diabetes odds, but these associations did not reach statistical significance (p > 0.05).
Conclusion. The healthy immigrant advantage extended to diabetes among US blacks, a finding that is explained by lower levels of overweight/obesity among the foreign-born compared to the US-born. Nonetheless, more than 71.4% of the foreign-born were overweight or obese. Understanding the mechanisms through which exposure to the US environment leads to higher obesity and diabetes risk may aid prevention efforts for the rapidly growing foreign-born black subpopulation.
Abstract Anticoagulation for atrial fibrillation has become more complex due to the introduction of new anticoagulant agents, the number and kinds of patients requiring therapy, and the interactions ...of those patients in the matrix of care. The management of anticoagulation has become a “team sport” involving multiple specialties in multiple sites of care. The American College of Cardiology, through the College’s Anticoagulation Initiative, convened a roundtable of experts from multiple specialties to discuss topics important to the management of patients requiring anticoagulation and to make expert recommendations on issues such as the initiation and interruption of anticoagulation, quality of anticoagulation care, management of major and minor bleeding, and treatment of special populations. The attendees continued to work toward consensus on these topics, and present the key findings of this roundtable in a state-of- the-art review focusing on the practical aspects of anticoagulation care for the patient with atrial fibrillation.
Objectives
Outcomes of community–acquired pneumonia (CAP) among HIV‐infected older adults are unclear.
Methods
Associations between HIV infection and three CAP outcomes (30‐day mortality, readmission ...within 30 days post‐discharge, and hospital length of stay LOS) were examined in the Veterans Aging Cohort Study (VACS) of male Veterans, age ≥ 50 years, hospitalized for CAP from 10/1/2002 through 08/31/2010. Associations between the VACS Index and CAP outcomes were assessed in multivariable models.
Results
Among 117 557 Veterans (36 922 HIV‐infected and 80 635 uninfected), 1203 met our eligibility criteria. The 30‐day mortality rate was 5.3%, the mean LOS was 7.3 days, and 13.2% were readmitted within 30 days of discharge. In unadjusted analyses, there were no significant differences between HIV‐infected and uninfected participants regarding the three CAP outcomes (P > 0.2). A higher VACS Index was associated with increased 30‐day mortality, readmission, and LOS in both HIV‐infected and uninfected groups. Generic organ system components of the VACS Index were associated with adverse CAP outcomes; HIV‐specific components were not. Among HIV‐infected participants, those not on antiretroviral therapy (ART) had a higher 30‐day mortality (HR 2.94 95% CI 1.51, 5.72; P = 0.002) and a longer LOS (slope 2.69 days 95% CI 0.65, 4.73; P = 0.008), after accounting for VACS Index. Readmission was not associated with ART use (OR 1.12 95% CI 0.62, 2.00 P = 0.714).
Conclusion
Among HIV‐infected and uninfected older adults hospitalized for CAP, organ system components of the VACS Index were associated with adverse CAP outcomes. Among HIV‐infected individuals, ART was associated with decreased 30‐day mortality and LOS.
Preterm birth or low birth weight is the single largest cause of death in newborns, however this mortality can be reduced through newborn care interventions, including Kangaroo Mother Care (KMC). ...Previously, a multi-country randomized controlled trial, coordinated by the World Health Organization (WHO), reported a significant survival advantage with initiation of continuous KMC immediately after birth compared with initiation of continuous KMC a few days after birth when the baby is considered clinically stable. Whether the survival advantage would lead to higher rates of neurodevelopmental morbidities, or the immediate KMC will also have a beneficial effect on cognitive development also, has not been investigated. We therefore propose to test the hypothesis that low-birth-weight infants exposed to immediate KMC will have lower rates of neurodevelopmental impairment in comparison to traditional KMC-treated infants, by prospectively following up infants already enrolled in the immediate KMC trial for the first 2 years of life, and assessing their growth and neurodevelopment.
This prospective cohort study will enroll surviving neonates from the main WHO immediate KMC trial. The main trial as well as this follow-up study are being conducted in five low- and middle-income countries in South Asia and sub-Saharan Africa. The estimated sample size for comparison of the risk of neurodevelopmental impairment is a total of 2200 children. The primary outcome will include rates of cerebral palsy, hearing impairment, vision impairment, mental and motor development, and epilepsy and will be assessed by the age of 3 years. The analysis will be by intention to treat.
Immediate KMC can potentially reduce low-birth-weight-associated complications such as respiratory disease, hypothermia, hypoglycemia, and infection that can result in impaired neurocognitive development. Neuroprotection may also be mediated by improved physiological stabilization that may lead to better maturation of neural pathways, reduced risk of hypoxia, positive parental impact, improved sleep cycles, and improved stress responses. The present study will help in evaluating the overall impact of KMC by investigating the long-term effect on neurodevelopmental impairment in the survivors.
Clinical Trials Registry-India CTRI/2019/11/021899. Registered on 06 November 2019. Trials registration of parent trial: ACTRN12618001880235; Clinical Trials Registry-India: CTRI/2018/08/015369.
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