POSoligo software for in vitro gene synthesis.

Oligonucleotide synthesis is vital for molecular experiments. Bioinformatics has been employed to create various algorithmic tools for the in vitro synthesis of nucleotides. The main approach to synthesizing long-chain DNA molecules involves linking short-chain oligonucleotides through ligase chain reaction (LCR) and polymerase chain reaction (PCR). Short-chain DNA molecules have low mutation rates, while LCR requires complementary interfaces at both ends of the two nucleic acid molecules or may alter the conformation of the nucleotide chain, leading to termination of amplification. Therefore, molecular melting temperature, length, and specificity must be considered during experimental design. POSoligo is a specialized offline tool for nucleotide fragment synthesis. It optimizes the oligonucleotide length and specificity based on input single-stranded DNA, producing multiple contiguous long strands (COS) and short patch strands (POS) with complementary ends. This process ensures free 5'- and 3'-ends during oligonucleotide synthesis, preventing secondary structure formation and ensuring specific binding between COS and POS without relying on stabilizing the complementary strands based on Tm values. POSoligo was used to synthesize the linear RBD sequence of SARS-CoV-2 using only one DNA strand, several POSs for LCR ligation, and two pairs of primers for PCR amplification in a time- and cost-effective manner.

A Study of the Relationship Between the Severity of Posterior Capsular Opacification Detected by Objective Detection Techniques and Visual Acuity.

PURPOSE: To explore the severity of posterior capsule opacification (PCO) using objective detection techniques and its relationship with visual acuity. SETTING: The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China. DESIGN: Prospective cohort study. METHODS: All patients underwent slit-lamp examination, intraocular pressure measurement (IOP), best-corrected visual acuity (BCVA) before neodymium: yttrium aluminium garnet (Nd:YAG) laser capsulotomy, followed by examination after fully dilated, including IOLMaster 700, optical coherence tomography (OCT), Sirius anterior segment analysis system (Sirius), color fundus photography (CFP). Conducting BCVA and IOP post-treatment again. Recording the thickness and density of posterior capsule, color fundus photography quality (CFPQ) and OCT Signal Strength (OCTSS). Analysis using Spearman correlation analysis, heatmaps, and ROC curves. RESULTS: A total of 83 eyes in 78 patients were included in this study. Spearman correlation analysis revealed correlations between pre-treatment BCVA and IOLMaster 700 PCO thickness (MT), IOLMaster 700 cumulative effect (MCE), Sirius PCO thickness (ST), Sirius maximum density (SMD), Sirius cumulative effect (SCE), OCTSS, and CFPQ (correlation coefficients were 0.500, 0.484, 0.465, -0.256, 0.317, -0.442, -0.412, all P＜0.05). The improvement of Vision Acuity (ImpVA) showed correlations with MT, MCE, ST, SCE, OCTSS, and CFPQ (correlation coefficients were -0.452, -0.471, -0.346, -0.278, 0.320, 0.381, all P＜0.05). For ImpVA, the predictive ability of IOLMaster 700 was superior to Sirius, and the joint model was significantly better than single factors. CONCLUSIONS: Posterior capsule thickness and cumulative effect were reliable indicators for evaluating PCO. Compared to Sirius, IOLMaster 700 demonstrates superior predictive ability and higher correlation.

Serum lymphocytes and cytokines: diagnostic value and influence on the immune status in patients with pulmonary tuberculosis.

OBJECTIVE: To determine the absolute number of serum T lymphocytes and cytokine levels and the characteristics of patients with active pulmonary tuberculosis and to assess their effect on the immune status of these patients and their diagnostic and predictive value for tuberculosis. METHODS: We included 1,069 patients with active tuberculosis, 51 patients with latent tuberculosis infection, and 600 health individuals. Absolute serum T-lymphocyte counts and cytokine levels were quantified. RESULTS: T lymphocytes were significantly reduced in patients with active tuberculosis when compared with healthy individuals. The immune function of patients gradually decreased with age and was stronger in female patients than in males. Th1 cells expressed higher levels of cytokines than did Th2 cells. Logistic regression analysis showed that reduced CD3+ T, CD8+ T, and NK cell counts, as well as reduced IL-4 and IFN-g expression, were independent influencing factors for active tuberculosis. ROC analysis showed that the sensitivity and specificity of absolute CD3+ T and CD8+ T lymphocyte counts and combined factors were significantly higher than were those of IL-4 and IFN-g for diagnosing active tuberculosis. CONCLUSIONS: Serum T-lymphocyte counts and cytokine levels can assess the immune status of tuberculosis patients; they are also useful biomarkers for predicting and diagnosing tuberculosis.

Serum lymphocytes and cytokines: diagnostic value and influence on the immune status in patients with pulmonary tuberculosis / Linfócitos e citocinas séricas: valor diagnóstico e influência no estado imunológico de pacientes com tuberculose pulmonar

ABSTRACT Objective: To determine the absolute number of serum T lymphocytes and cytokine levels and the characteristics of patients with active pulmonary tuberculosis and to assess their effect on the immune status of these patients and their diagnostic and predictive value for tuberculosis. Methods: We included 1,069 patients with active tuberculosis, 51 patients with latent tuberculosis infection, and 600 health individuals. Absolute serum T-lymphocyte counts and cytokine levels were quantified. Results: T lymphocytes were significantly reduced in patients with active tuberculosis when compared with healthy individuals. The immune function of patients gradually decreased with age and was stronger in female patients than in males. Th1 cells expressed higher levels of cytokines than did Th2 cells. Logistic regression analysis showed that reduced CD3+ T, CD8+ T, and NK cell counts, as well as reduced IL-4 and IFN-g expression, were independent influencing factors for active tuberculosis. ROC analysis showed that the sensitivity and specificity of absolute CD3+ T and CD8+ T lymphocyte counts and combined factors were significantly higher than were those of IL-4 and IFN-g for diagnosing active tuberculosis. Conclusions: Serum T-lymphocyte counts and cytokine levels can assess the immune status of tuberculosis patients; they are also useful biomarkers for predicting and diagnosing tuberculosis.

RESUMO Objetivo: Determinar o número absoluto de linfócitos T séricos e os níveis de citocinas séricas, bem como as características, de pacientes com tuberculose pulmonar ativa e avaliar o efeito desses no estado imunológico desses pacientes e seu valor diagnóstico e preditivo para tuberculose. Métodos: Foram incluídos 1.069 pacientes com tuberculose ativa, 51 pacientes com tuberculose latente e 600 indivíduos saudáveis. Foram realizadas a contagem absoluta de linfócitos T séricos e a quantificação de citocinas séricas. Resultados: Os linfócitos T estavam significativamente reduzidos nos pacientes com tuberculose ativa em comparação com os indivíduos saudáveis. A função imunológica dos pacientes diminuiu gradativamente com a idade e mostrou-se mais forte nas mulheres do que nos homens. As células Th1 expressaram maiores níveis de citocinas do que as células Th2. A análise de regressão logística mostrou que contagens reduzidas de células T CD3+, T CD8+ e NK e expressão reduzida de IL-4 e IFN-g foram fatores de influência independentes para tuberculose ativa. A análise ROC mostrou que a sensibilidade e especificidade dos valores absolutos de linfócitos T CD3+ e T CD8+ e de fatores combinados foram significativamente maiores do que as da IL-4 e do IFN-g para o diagnóstico da tuberculose ativa. Conclusões: A contagem de linfócitos T séricos e os níveis de citocinas séricas podem avaliar o estado imunológico de pacientes com tuberculose; também são biomarcadores úteis na predição e diagnóstico da tuberculose.

Laser-induced choroidal neovascularization: A case report and some reflection on animal models for age-related macular degeneration.

RATIONALE: Laser induced maculopathy includes retinal photoreceptor disruption, macular hole, macular hemorrhage, and rarely choroidal neovascularization (CNV). Here we report a case of laser induced CNV that was treated by intravitreal anti-vascular endothelial growth factor (VEGF) injection and resulted in visual improvement and CNV resolution during 1-year follow up. In addition, the case of laser induced CNV treated with intravitreal anti-VEGF injections are reviewed for the first time in literature. PATIENT CONCERNS: A 7-year-old boy presented to our department with blurred vision in his right eye for 2 months. The symptom immediately happened after the boy staring at the laser beam for a few seconds. Examination of ocular fundus with slit lamp showed yellowish lesion in macula in his right eye. DIAGNOSES: CNV was confirmed by fundus examinations, including color fundus photograph, spectral domain optical coherence tomography, fluorescein angiography, and spectral domain optical coherence tomography angiography. INTERVENTIONS: After the diagnosis of laser induced CNV, intravitreal ranibizumab (LUCENTIS, NOVARTIS) injection was performed. OUTCOMES: After 1 injection of intravitreal ranibizumab, the best corrected visual acuity improved from 20/50 to 30/50 and CNV gradually regressed during 1-year follow up. LESSONS: For young patients with laser induced CNV, intravitreal anti-VEGF injections may be helpful in visual improvement and CNV regression. Moreover, age seems to be a significant factor thus we propose that old animals may be more appropriate for laser induced CNV animal models of age-related macular degeneration.

Influence of measurement differences of anterior chamber depth and axial length on lens thickness evaluation in cataract patients: a comparison of two tests.

BACKGROUND: The purpose of this study was to investigate the agreement of lens thickness (LT) measurements made by contact A-scan ultrasonography and Lenstar LS900 as well as the influence of anterior chamber depth (ACD) and axial length (AL) measurement differences on LT measurement in cataract patients in the two techniques. METHODS: 1247 cataract patients (1247 eyes) participated in this retrospective cross-sectional study. Ocular biometric measurements were performed with Lenstar LS900 and A-scan ultrasonography respectively, and the measured results of AL, ACD and LT were compared using Pearson correlation coefficients (r) and Bland-Altman analyses. RESULTS: Bland-Altman analyses showed poor agreement between the A-scan ultrasonography and Lenstar LS900 in measuring AL and ACD. The average difference of LT was 0.01 mm; the consistency limit was - 0.86 mm, 0.88 mm; and 95.27% of datapoints were within the 95% consistency limit. The consistency of LT measurements between the two techniques was poor for those subjects whose ACD or AL values were beyond the 95% consistency limit. Among the subjects whose AL or ACD values measured by A-scan ultrasonography were greater than those measured by Lenstar LS900, 93.33% of them were within the 95% consistency limit, suggesting that the consistency of LT measurement between the two techniques was poor. Of patients whose ACD or AL measured by A-scan ultrasonography were smaller than that of Lenstar LS900, 96.01% of them were within the 95% consistency limit. CONCLUSIONS: There was good agreement of the LT measurements between A-scan ultrasonography and Lenstar LS900, except for the axis deviating from the apparent axis during A-scan ultrasonography. If this error can be avoided, A-scan ultrasonography can replace Lenstar LS900 in LT measurement in cataract patients.

The clinical study on treatment of CD19-directed chimeric antigen receptor-modified T cells in a case of refractory Richter syndrome.

Richter syndrome (RS) indicates the transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma (mostly DLBCL). Richter syndrome is a rare complication with an aggressive clinical course, bearing an unfavorable prognosis. Currently, there is no effective treatment for it. As a novel cellular-based immune therapy, chimeric antigen receptor-modified T (CART) cells treatment is gradually used in treating hematological malignancies, especially in CD19+ B-cell malignancy. Therefore, CD19-directed chimeric antigen receptor-modified T cells (CART-19) treatment is promising to be used as a new method for RS patients. In our study, one RS patient expressing high level of CD19 molecule was enrolled in clinical trial; he has received a series of treatments but did not achieve a satisfactory therapeutic effect. The patient totally received 3.55 × 108 autologous CART-19 cells infusion. After CART-19 infusion, the mainly clinical side effect was repeated fever. The maximal duration period was 24 days and the highest temperature was 40.1°C. Pancytopenia and significantly serum cytokines level rise were observed, including IFN-γ, IL-6, and IL-10. Before discharge, the level of cytokines reduced to normal levels. In addition, we detected the serum biochemical indices as like K+ , Ca2+ , creatinine, and glutamic-pyruvic transaminase, all of these indices were normal. This showed that there was no tumor necrosis syndrome after treatment. The proportion of B cells in patient's peripheral blood decreased from 72% to 40.2% after infusion, co-occurring with reduction in lymph nodes and hematopoietic reconstitution. Based on the recent revolution in the therapeutic landscape for hematological malignancies including B-cell lymphomas, CART-CD19 cell therapy as a new therapeutic option for RS might be available in the coming years. It aims to reduce patient's tumor burden, prolong their survival time, and provide opportunities for other sequential therapy such as chemotherapy and bone marrow transplantation.

Development of Inducible CD19-CAR T Cells with a Tet-On System for Controlled Activity and Enhanced Clinical Safety.

The tetracycline regulatory system has been widely used to control the transgene expression. With this powerful tool, it might be possible to effectively control the functional activity of chimeric antigen receptor (CAR) T cells and manage the severe side effects after infusion. In this study, we developed novel inducible CD19CAR (iCAR19) T cells by incorporating a one-vector Tet-on system into the CD19CAR construct. The iCAR19 T cells showed dox-dependent cell proliferation, cytokine production, CAR expression, and strong CD19-specific cytotoxicity. After 48 h of dox induction, the relative CAR expression of induced cells was five times greater than that of uninduced cells. Twenty-four hours after dox removal, CAR expression significantly decreased by more than 60%. In cytotoxicity assays, dox-treated cells induced significantly higher specific lysis against target cells. These results suggested that the activity of iCAR19 T cells was successfully controlled by our Tet-on system, offering an enhanced safety profile while maintaining a robust anti-tumor effect. Besides, all manufacture processes of the lentiviral vectors and the T cells were conducted according to the Good Manufacturing Practice (GMP) standards for subsequent clinical translation.

Adipose-derived stem cells in articular cartilage regeneration: current concepts and optimization strategies.

Knee osteoarthritis (KOA) is the most common progressive joint disorder associated with disability in the world. As a chronic disease, KOA has multifactorial etiology. However, the poor self-healing ability of the articular cartilage due to its intrinsic tissue hypovascularity and hypocellularity seems to be directly incriminated in the physio-pathological mechanism of KOA. While conventional therapies result in unfavorable clinical outcomes, regenerative cell therapies have shown great promise in articular cartilage regeneration. Adipose-derived stem cells (ASCs) appear to be an ideal alternative to bone-marrow derived stem cells (BMSCs) and autologous chondrocytes, due to their lower immunogenicity, richer source and easier acquisition. Since the first case report in 2011, ASCs have demonstrated safety and efficacy for articular cartilage regeneration in several phase I/II clinical trials. However, different levels of abnormality were found in the regenerated cartilage for most of the patients. A large portion of recent publications investigated different optimization strategies to improve the therapeutic function of ASCs, including cell source selection, preconditioning and co-delivery. Herein, we give an update on the latest research progress on ASCs, with a focus on the most promising optimization strategies for ASC-based therapy.

Tumor-targeting TRAIL expression mediated by miRNA response elements suppressed growth of uveal melanoma cells.

Malignant uveal melanoma severely damages eye function and is prone to metastasize to other organs. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent to treat uveal melanoma because of its induction of apoptosis in cancer cells both at primary and metastatic sites. However, TRAIL therapy lacks tumor specificity in the current delivery systems for uveal melanoma treatment, thereby causing cytotoxiciy to normal tissues. To improve uveal melanoma specificity of adenovirus-based TRAIL introduction, we used miRNA response elements (MREs) of miR-34a, miR-137 and miR-182, which have been shown to have reduced expression in uveal melanoma cells, to regulate its expression. miR-34a, miR-137 and miR-182 all had lower expression levels in uveal melanoma cell lines, compared with normal cells. MREs-regulated luciferase activity was reduced in normal cell lines, but not significantly attenuated in uveal melanoma cells. The infection of MRE-regulated TRAIL-expressing adenoviral vector (Ad-TRAIL-3MREs) led to high level of TRAIL expression in uveal melanoma cell lines, but not in normal cells. Strong expression of TRAIL had a high anti-tumor capacity by inducing apoptosis in uveal melanoma cells. In contrast, Ad-TRAIL-3MREs had no cytotoxicity to normal cell lines. Animal experiments further confirmed tumor-suppressing effect of Ad-TRAIL-3MREs on uveal melanoma xenografts and its biosafety to hepatic tissues. Collectively, we constructed an MRE-directed TRAIL-expressing adenoviral vector and provided evidence that this vector possessed high anti-tumor activity and uveal melanoma specificity.