Recombinant protein-based SARS-CoV-2 vaccines are needed to fill the vaccine equity gap. Because protein-subunit based vaccines are easier and cheaper to produce and do not require special ...storage/transportation conditions, they are suitable for low-/middle-income countries. Here, we report our vaccine development studies with the receptor binding domain of the SARS-CoV-2 Delta Plus strain (RBD-DP) which caused increased hospitalizations compared to other variants. First, we expressed RBD-DP in the Pichia pastoris yeast system and upscaled it to a 5-L fermenter for production. After three-step purification, we obtained RBD-DP with > 95% purity from a protein yield of > 1 g/L of supernatant. Several biophysical and biochemical characterizations were performed to confirm its identity, stability, and functionality. Then, it was formulated in different contents with Alum and CpG for mice immunization. After three doses of immunization, IgG titers from sera reached to > 10
and most importantly it showed high T-cell responses which are required for an effective vaccine to prevent severe COVID-19 disease. A live neutralization test was performed with both the Wuhan strain (B.1.1.7) and Delta strain (B.1.617.2) and it showed high neutralization antibody content for both strains. A challenge study with SARS-CoV-2 infected K18-hACE2 transgenic mice showed good immunoprotective activity with no viruses in the lungs and no lung inflammation for all immunized mice.
Selective autophagy of the endoplasmic reticulum (ER), namely ER-phagy, is mediated by ER-localized receptors, which are recognized and sequestered by GABARAP/LC3B-decorated phagophores and ...transferred to lysosomes for degradation. Being one such receptor,
plays critical roles in cellular processes such as protein quality control and neuronal survival.
has also been associated with different cancers, although its exact role remains elusive. We report here that the
gene encodes not one but at least two different protein isoforms: the full-length and the NH
terminally truncated forms. Their relative expression shows extreme variation, both within normal tissues and among cancer types. Expression of full-length FAM134B is restricted to the brain, testis, spleen, and prostate. In contrast, NH
terminally truncated FAM134B is dominant in the heart, skeletal muscle, kidney, pancreas, and liver. We compared wild-type and knockout mice to study the role of the
gene in starvation. NH
terminally truncated FAM134B-2 was induced in the liver, skeletal muscle, and heart but not in the pancreas and stomach following starvation. Upon starvation,
mice differed from wild-type mice by less weight loss and less hyperaminoacidemic and hypocalcemic response but increased levels of serum albumin, total serum proteins, and α-amylase. Interestingly, either NH
terminally truncated
or both isoforms were downregulated in liver, lung, and colon cancers. In contrast, upregulation was observed in stomach and chromophobe kidney cancers.
We reported tissues expressing FAM134B-2 such as the kidney, muscle, heart, and pancreas, some of which exhibit stimulated expression upon nutrient starvation. We also demonstrated the effect of
deletion during ad libitum and starvation conditions. Resistance to weight loss and hypocalcemia, accompanied by an increase in serum albumin and α-amylase levels, indicate critical roles of
in physiology. Furthermore, the differential expression of FAM134B isoforms was shown to be significantly dysregulated in human cancers.
Construction of synthetic genes is today the most elegant way to optimize the heterologous expression of a recombinant protein. The availability of sequences of entire genome has significantly ...increased the number of protein targets which many of them will need to be overexpressed in cells other than the original source of DNA. Gene synthesis often provides a fast and economically efficient approach. In this study we have optimized a two-step polymerase chain reaction (2-step PCR) method for the fast and extremely accurate synthesis of a 186 bp CUP1 gene encoding yeast Saccharomyces cerevisiae copper metallothionein. A total of the six overlapping oligonucleotides ranged from 43 to 49 in length, designed with the unique restriction sites, were assembled in a single step PCR. The assembly was then further amplified by second PCR to produce a synthetic gene which has been cloned into the pET28a(+) vector to allow the expression of CUP1 gene in E. coli BL21 (DE3) host cell. In order to compare the difference in expression level of the gene with optimized codon usage for E. coli, CUP1 gene was redesigned according to codon bias of host cell. A significant increase of expression level of codon optimized gene was obtained compared to original sequence of CUP1 gene of copper metallothionein in yeast Saccharomyces cerevisiae.
Objective: In this study, it is aimed to constitute a chronic toxoplasmosis model using toxoplasma gondii (T. gondii) ME49 strain that is supposed to produce tissue cysts, to compare healthy and ...infected mice in terms of behavioral changes. In addition, the relationship between behavioral changes and brain lesions was questioned. Method: Before experimental application, out of a total of twenty-one 2-month- old Swiss albino mice, T. gondiantibody-free, 14 were infected by intraperitoneal (IP) (n=8) or oral (n=6) inoculation of 2x102 Toxoplasma gondii ME49 oocysts. Four months later, fear- and anxiety-related behavioral changes in infected and healthy control groups were comparatively evaluated with plus-maze test. At the end of the experiment (45 day after inoculation), euthanasia was carried out on all mice and their brains were examined histopathologically and immunohistochemically for the presence of T.gondii tissue cysts. Results: Infected mice had low levels of anxiety, they have enteredthe open arms more frequently and have spent more time in the open arms of the maze compared to controls. Pathologically, in the infected group, high rates of gliosis, perivascular cell infiltration, meningitis, and neuron necrosis were observed especially in the parietal and temporal lobes, cornu ammonis, amygdala, and thalamus compared to the other parts of the brain. A higher number of tissue cyst formations were positively correlated with the lesion severity in these parts of the brain. Conclusion: It can be suggested that, tissue cysts and neuropathological changes in chronically infected mice brains have direct concern with behavioral manipulation which results from low anxiety levels. (Archives of Neuropsychiatry 2012; 49:139-144) Key words: Toxoplasma gondii, mice, tissue cyst, behavior Amac: Bu calismada; kist olusturan T. gondii ME 49 susu ile enfekte farelerde olusan anksiyete ile iliskili davranis degisiklikleri ve beyin lezyonlariyla iliskisinin arastirilmasi amaclanmistir. Yontem: Uygulama oncesi serumlarinda anti-T. gondii antikorlarinin olmadigi gosterilen 2 aylik 21 adet erkek Swiss albino fareden deney grubundaki 14'une; intraperitoneal (IP) (n=8) ve oral (n=6) yollarla 2x102 Toxoplasma gondii ME49 ookisti verildikten 4 hafta sonra, anksiyete veya korku ile iliskili davranislari T. gondii ile enfekte fareler ile kontrol grubu saglikli farelerde karsilastirmali olarak degerlendirildi. Deney bitiminde (inokulasyon sonrasi 45'inci gun) farelere otenazi yapilarak, beyinleri histopatolojik olarak ve T. gondiidoku kisti antijeni yonunden immunoperoksidaz test ile incelendi. Bulgular: T. gondii ile enfekte farelerin daha dusuk duzeyde anksiyeteye sahip olduklarini, enfekte olmayan kontrol grubundaki farelere gore daha yuksek oran ve sureyle acik kolu tercih ettiklerini ortaya koydu. Patolojik olarak; enfekte grupta ozellikle parietal ve temporal loblar, kornu ammonis, amigdala, talamus ve cevresinde, beynin diger bolumlerine oranla daha yuksek derecede gliozis, perivaskuler hucre infiltrasyonu, meningitis ve noron nekrozlari gozlendi. Beyinde T. gondii doku kisti yerlesimi de anilan lezyon siddeti ile dogru orantili olarak diger alanlara oranla daha yuksek sayidaydi. Sonuc: Kronik toksoplazmozlu farelerin beyinlerinde sekillenen T.gondiidoku kistleri ve noropatolojik degisikliklerin dogrudan anksiyetenin azaltilmasina yonelik davranis manipulasyonu ile iliskili oldugu one surulebilir. (Noropsikiyatri Arsivi 2012; 49:139-144) Anahtar kelim eler: Toxoplasma gondii, fare, doku kisti, davranis
•Experimental values for CD, spray angle and film thickness of a simplex nozzle is obtained using water at four different flow rates.•2D axisymmetric and full sector 3D VOF simulations are carried ...out using IDDES turbulence model.•Full benchmark of 2D and 3D methods are done for the accuracy in validation parameters along with the computational cost.•3D VOF method can provide highly accurate results while 2D method predictions may be used to obtain a general outlook of the flow field with a low cost.•If the inlet slot flow separation behavior is known, 2D method can yield quite satisfying results for the estimation of CD, spray angle and film thickness.
In this study, a simplex nozzle is tested with water for the benchmarking of different flow simulation models. A large scale Plexi-glass transparent nozzle is used to reduce the influence of production tolerances on the performance. Experiments are conducted at different flow rates and CD, spray angle and film thickness parameters are evaluated. 2D and 3D hybrid RANS-LES multiphase flow simulations of simplex nozzle are validated against the experimental data. Multiphase nature of the flow is modelled by volume of fluid method. The main goal is to assess the capabilities and drawbacks of 2D axisymmetric and full sector 3D modeling approaches. It is observed that although full sector 3D simulations require HPC cluster systems, accuracies in validation parameters are quite satisfying. Conversely, 2D axisymmetric simulations which can be run on a single core and give a general outlook of the flow field, they show an overshoot of CD and film thickness over the selected range of flow rate. It is shown that this overshoot is mostly related with the inlet boundary condition, which can not take the flow contraction and/or separation at the inlet slots into account. After correcting the inlet velocity 2D simulations by using the 3D results, it is shown that the predictions can be quite close to the experimental data.
Covalent protein kinase inhibitors exploit currently noncatalytic cysteines in the adenosine 5'-triphosphate (ATP)-binding site via electrophiles directly appended to a reversible-inhibitor scaffold. ...Here, we delineate a path to target solvent-exposed cysteines at a distance >10 Å from an ATP-site-directed core module and produce potent covalent phosphoinositide 3-kinase α (PI3Kα) inhibitors. First, reactive warheads are used to reach out to Cys862 on PI3Kα, and second, enones are replaced with druglike warheads while linkers are optimized. The systematic investigation of intrinsic warhead reactivity (
), rate of covalent bond formation and proximity (
and reaction space volume
), and integration of structure data, kinetic and structural modeling, led to the guided identification of high-quality, covalent chemical probes. A novel stochastic approach provided direct access to the calculation of overall reaction rates as a function of
,
,
, and
, which was validated with compounds with varied linker lengths. X-ray crystallography, protein mass spectrometry (MS), and NanoBRET assays confirmed covalent bond formation of the acrylamide warhead and Cys862. In rat liver microsomes, compounds
and
outperformed the rapidly metabolized CNX-1351, the only known PI3Kα irreversible inhibitor. Washout experiments in cancer cell lines with mutated, constitutively activated PI3Kα showed a long-lasting inhibition of PI3Kα. In SKOV3 cells, compounds
and
revealed PI3Kβ-dependent signaling, which was sensitive to TGX221. Compounds
and
thus qualify as specific chemical probes to explore PI3Kα-selective signaling branches. The proposed approach is generally suited to develop covalent tools targeting distal, unexplored Cys residues in biologically active enzymes.
•Female patients with peripheral PsA have lower remission and MDA rates and higher levels of disease activity than males.•Female patients experience a more severe course of PsA, with higher levels of ...pain and fatigue, lower quality of life, and increased functional limitations.•The predictors of MDA are similar between genders in patients with peripheral PsA.
This study sought to compare disease activity, clinical features, and patient-reported outcomes concerning anxiety, depression, fatigue, function, quality of life, and fibromyalgia between female and male patients with peripheral PsA in a Turkish population.
This multi-center Turkish League Against Rheumatism (TLAR) Network study included 1038 patients (678 females, 360 males) diagnosed with peripheral PsA according to the CASPAR criteria. The demographic and clinic parameters of the patients were recorded. Disease activity was evaluated using the scores of DAS28 and cDAPSA. Remission, minimal disease activity (MDA), and very low disease activity (VLDA) were determined. Health Assessment Questionnaire (HAQ), Short-Form-36 (SF-36), Hospital Anxiety and Depression Scale (HAD), fatigue VAS (0–10), and Fibromyalgia Rapid ScreeningTool (FiRST) were used. Disease activity and patient-reported outcomes were compared in male and female patients, and the predictors of MDA for both genders were analyzed.
The patients’ mean age was 47.6years (SD: 12) for females and 46.3years (SD: 12.3) for males. In terms of DAS28 and cDAPSA, female patients had significantly higher disease activity scores, while male patients had significantly higher remission rates (P<0.05). There was a significant difference in the rate of MDA in favor of males (P<0.05), but not in VLDA. The incidences of dactylitis, enthesitis, tenosynovitis, and inflammatory bowel disease were similar in male and female patients, except for spondylitis, which was higher in males (P<0.05). Overall, although there was no significant between-group difference in age and disease duration, female patients had significantly higher BMI and late-onset disease (P<0.05). Female patients had higher HAD, HAQ, and FiRST and lower SF-36 scores than males (P<0.05). In both male and female patients, the disease activity score of cDAPSA was significantly correlated with the scores of FiRST, HAD, VAS-F, and HAQ (P<0.05). In regression analysis, tender joint count, swollen joint count, PASI, pain VAS, and enthesitis were the MDA predictors in both genders.
In patients with peripheral PsA, males are more likely to develop spondylitis while other extraarticular manifestations are similar. Female patients appear to have lower rates of remission and MDA and higher levels of disease activity. Female patients experience a more severe course of PsA, with higher levels of pain and fatigue, lower quality of life, and increased functional limitations. The predictors of MDA, i.e., tender joint count, swollen joint count, PASI, pain VAS, and enthesitis are similar between the two genders.
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