The spliceosome removes introns from messenger RNA precursors (pre-mRNA). Decades of biochemistry and genetics combined with recent structural studies of the spliceosome have produced a detailed view of the mechanism of splicing. In this review, we aim to make this mechanism understandable and provide several videos of the spliceosome in action to illustrate the intricate choreography of splicing. The U1 and U2 small nuclear ribonucleoproteins (snRNPs) mark an intron and recruit the U4/U6.U5 tri-snRNP. Transfer of the 5' splice site (5'SS) from U1 to U6 snRNA triggers unwinding of U6 snRNA from U4 snRNA. U6 folds with U2 snRNA into an RNA-based active site that positions the 5'SS at two catalytic metal ions. The branch point (BP) adenosine attacks the 5'SS, producing a free 5' exon. Removal of the BP adenosine from the active site allows the 3'SS to bind, so that the 5' exon attacks the 3'SS to produce mature mRNA and an excised lariat intron. Expected final online publication date for the Annual Review of Biochemistry, Volume 89 is June 22, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Michal Ješeta, Jana Antalíková, Adéla Doubravská
et al.
The separation of human spermatozoa is an important step in therapy of human
infertility. Given that male fertility is decreasing and, as a result,
fertilization techniques based on microinjection of sperm into the cytoplasm are
being used more intensively, this question is becoming increasingly relevant. In
recent years, microfluidic sperm processing techniques have been increasingly
used. These methods are simple and easy to use, however, the question is to what
extent they select the correct sperm. They are essentially based mainly on
motility and do not reflect other navigational approaches such as chemotaxis,
thermotaxis or rheotaxis. This review compares traditional, advances and
novel in vitro methods of sperm separation which are commonly used
during human infertility therapy in context of in vivo sperm separation
in female reproductive system.
IntroductionChronic nonhealing wounds are one of the most serious complications of diabetes mellitus (DM), with limited treatment options. Hydrogen sulfide (H2S) plays a protective role against multiple inflammatory diseases. This study aimed to explore the effects of H2S on diabetic skin wound healing and its underlying mechanisms.MethodsA streptozotocin-induced diabetic rat model was established, and the rats were randomly divided into control, DM, and DM + NaHS (a donor of H2S) groups. Full-thickness wounds were made on the dorsal skin of the rats. H2S levels and H2S-synthesizing enzyme expression were evaluated in the wound tissue. Wound healing, histological changes, inflammasome activation, fibroblast pyroptosis, and phosphorylation of signaling components of nuclear factor kappa B (NF-κB) pathway were assessed.ResultsThe results showed that NaHS administration effectively restored H2S levels and promoted skin wound healing, as evidenced by the amelioration of histological changes and increased collagen deposition in diabetic rats. Meanwhile, NaHS treatment inhibited macrophage M1 polarization and decreased the levels of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in diabetic wound tissues, notably, suppressing NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation and fibroblast pyroptosis. In addition, NaHS treatment was able to inhibit the activation of NF-κB pathway in the wound tissues.ConclusionTaken together, these results show that H2S promotes skin wound healing in diabetic rats and may be involved in the restoration of H2S levels, inhibition of NLRP3 inflammasome activation, and fibroblast pyroptosis, suggesting that it may be a promising therapeutic agent for treating diabetic skin wounds.
Imene Djaalab, Samia Haffaf, Hadria Mansour-Djaalab
et al.
Animal Welfare has a significant impact on the dairy cow’s health, behaviour, productivity and milk quality. By implementing husbandry practices that respect the physical, behavioural and emotional needs of dairy cows, the dairy industry can improve the sustainability of its operations and meet rising expectations. The aim of this study is to evaluate the impact of housing systems (free vs. tied) on dairy cow health through musculoskeletal health indicators and lameness scores. The hypothesis that dairy cows reared in free housing have a better quality of health than cows reared in restrained housing is tested. Thus, 300 dairy cows of the Holstein and Montbeliarde breeds were selected from dairy farms in five municipalities of Constantine province (eastern Algeria). The results showed that the frequency of severe lameness did not exceed 12% in stalls with restraints and more than 42% of light lameness are in free-stall housing (<i>p</i> < 0.001). These results reflect a lack of comfort in restricted housing, with an impact on dairy performances. Moreover, the monitoring of lame cows and the functional trimming of their hooves should be frequent. It is also important to implement a cull policy for unproductive cows. Finally, it is very important to provide adequate training to farmers in order to improve the well-being of dairy cows.
ABSTRACT Background Isoproterenol (ISO) is a nonselective beta‐adrenergic receptor agonist known for its vasodilatory effects. This experiment aims to investigate whether intrauterine ISO administration could alter vascular indices and follicular development in postpartum Holstein cows. Objectives The objectives are to evaluate the effects of intrauterine ISO administration on vascular changes and its impact on follicular development compared to placebo groups. Study Design This randomized controlled study was conducted on 36 Holstein cows selected based on their health status, including only those free from reproductive, metabolic and infectious disorders. Methods The cows (n = 36) were divided into two groups as control received distilled water alone (CON, n = 18) and experiment received 4 mg ISO in 40 mL distilled water (ISO, n = 18) and four subgroups as CON‐I (n = 9), CON‐II (n = 9), ISO‐I (n = 9) and ISO‐II (n = 9) according to days of intrauterine administration (I or II represents to 1 or 2 days after ovulation, respectively). Uterine and ovarian artery blood flows were assessed before and after administration by Doppler ultrasonography. Blood samples were collected both before and after administration (on Day 1 or 2) and on Days 3, 6 and 9 post‐ovulation for hormonal analysis. Antral follicle count (AFC) was recorded on the blood sampling days. Data were analysed via mixed model ANOVA. Results Intrauterine ISO administration significantly increased the pulse rate (PR) in the ovaries (89.4 vs. 65.5 bpm, p < 0.0001) and uterus (90.6 vs. 64.2 bpm, p < 0.0001). Early AFC (1–2.9 mm) decreased, whereas small AFC (3–4.9 mm) increased in the ISO groups. The weighted average antral follicle size (WAAFS) significantly increased in the ISO group but remained unchanged in the controls. Hormonal analysis revealed elevated levels of FSH (626 vs. 468 mIU/mL), AMH (61.3 vs. 46.4 ng/L), E2 (138 vs. 122 ng/L), P4 (15.3 vs. 10.6 ng/mL), IGF‐1 (62.6 vs. 25.1 ng/mL) and IGFBP‐3 (28.4 vs. 16.5 ng/mL) in the ISO groups (p < 0.0001). Conclusions The findings indicate that intrauterine administration of ISO on Day 1 post‐ovulation could be a promising ‘adjunct technique’ for future research focussed on minimizing dependence on exogenous hormones or improving the sensitivity of follicles to endogenous hormonal signals, thereby potentially enhancing oocyte yield.
Introduction: Climate change poses a significant global health threat impacting regions unevenly based on their vulnerabilities. India, with its distinct geography and climate, is particularly at risk. This study aimed to assess the knowledge, attitudes, and practices of healthcare workers regarding climate change and its impact on, human health, with a specific focus on the National Programme for Climate Change and Human Health. Methodology: This was a cross sectional study, conducted among healthcare workers in Datia, Madhya Pradesh. Healthcare workers at the Medical College and District Hospital in Datia were given a semi-structured questionnaire via Google Form between October 1 and October 30, 2023. The total eligible population for participation was approximately 200 healthcare workers, including teachers from the Medical College, PG residents, and medical officers from the District Hospital, Datia, Madhya Pradesh. Results: The analysis revealed that 62.5% of participants were aware of NPCCHH, with 52.4% understanding its objectives. Moreover, 79.2% displayed positive attitudes towards climate change, emphasizing the need for awareness about climate changes impact on vulnerable communities. While a minority (29.2%) engaged in climate change related awareness programs and 20% monitored air quality index (AQI) daily, the majority (70%) practiced eco-friendly activities like recycling, conserving water, and using energy efficient appliances daily. Conclusion: Lower knowledge levels among postgraduate residents highlight the need for targeted educational initiatives. Finding of positive attitude among all healthcare worker groups signal encouraging environment for future climate change interventions. The variation in practices among groups suggests a need for formal training programs on heat-related illness and air pollution monitoring.
Muhammad Mazher Irshad , Kausar Noor, Muhammad Imran Khan
et al.
Background: Laparoscopic surgery is increasingly preferred for various abdominal procedures due to its minimally invasive nature. However, port site complications, particularly wound infections, remain a significant concern. The method of umbilical access—either intra-umbilical or periumbilical—may influence the rate of postoperative infections, yet evidence comparing the two remains limited. The study aimed to compare the frequency of wound infection between intra-umbilical and periumbilical incisions in patients undergoing laparoscopic appendectomy or cholecystectomy.
Methods: A descriptive study was conducted over six months in the Department of Surgery at Khyber Teaching Hospital, Peshawar. A total of 201 patients undergoing laparoscopic surgeries were enrolled using a non-probability consecutive sampling technique. Patients were divided into Group A (intra-umbilical incision, n=101) and Group B (periumbilical incision, n=100). Baseline demographics, comorbidities, and type of surgery were recorded. Postoperative wound infections were assessed within two weeks based on predefined clinical criteria. Data were analyzed using SPSS v25, with Chi-square and Fisher's exact tests applied where appropriate.
Results: The overall wound infection rate was 11.4%, with 8 infections (7.9%) in Group A and 15 infections (15%) in Group B. The infection rate was nearly double in the periumbilical group compared to the intra-umbilical group. Other variables, including comorbidities and type of surgery, were comparable between the groups.
Conclusion: Intra-umbilical incisions were associated with a lower incidence of wound infections compared to periumbilical incisions in laparoscopic procedures. This method may offer a safer and cosmetically superior alternative for initial port access in routine laparoscopic surgeries
Alinny R. Isaac, Mariana G. Chauvet, Ricardo Lima-Filho
et al.
Abstract Major depressive disorder (MDD) is a significant cause of disability in adults worldwide. However, the underlying causes and mechanisms of MDD are not fully understood, and many patients are refractory to available therapeutic options. Impaired control of brain mRNA translation underlies several neurodevelopmental and neurodegenerative conditions, including autism spectrum disorders and Alzheimer’s disease (AD). Nonetheless, a potential role for mechanisms associated with impaired translational control in depressive-like behavior remains elusive. A key pathway controlling translation initiation relies on the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α-P) which, in turn, blocks the guanine exchange factor activity of eIF2B, thereby reducing global translation rates. Here we report that the expression of EIF2B5 (which codes for eIF2Bε, the catalytic subunit of eIF2B) is reduced in postmortem MDD prefrontal cortex from two distinct human cohorts and in the frontal cortex of social isolation-induced depressive-like behavior model mice. Further, pharmacological treatment with anisomycin or with salubrinal, an inhibitor of the eIF2α phosphatase GADD34, induces depressive-like behavior in adult C57BL/6J mice. Salubrinal-induced depressive-like behavior is blocked by ISRIB, a compound that directly activates eIF2B regardless of the phosphorylation status of eIF2α, suggesting that increased eIF2α-P promotes depressive-like states. Taken together, our results suggest that impaired eIF2-associated translational control may participate in the pathophysiology of MDD, and underscore eIF2-eIF2B translational axis as a potential target for the development of novel approaches for MDD and related mood disorders.
Molecular biology plays an important role in understanding the structures, functions and internal controls within each cell. All this can be used to diagnose the diseases, effectively target new medicines and better understand cellular physiology. The bacteria that cause Lyme disease are difficult to observe directly in body tissues and too time-consuming to grow in the laboratory. Lyme disease can affect several body systems and produce a broad range of symptoms. Not everybody with Lyme disease has all the symptoms and many of the symptoms are not specific but may occur with other diseases. Common laboratory diagnostic methods have many false positive results in contaminated areas. These problems have made scientists think of finding accurate and fast methods to diagnose. The accuracy and precision of molecular biological methods have made an important field of research to identify the pathogen of this disease. Borrelia Burgdorferi is a species of the Spirochaeta order and Borrelia genus. This type of Gram-negative bacteria is the most important cause of Lyme disease. Five of these species, Borrelia afzeli, Borrelia garinii, Borrelia bavariansis, Borrelia bergdorferi senses strict, and Borrelia spirmani, have been described as causative agents of Lyme disease in humans. There are 36 known Borrelia species. In the Borrelia family, three species cause Lyme disease or borreliosis, the most important cause in USA is Borrelia Burgdorferi and the main cause in Europe are Borrelia afzelii and Borrelia garinii. Accurate molecular tests are designed for specific detection and isolation of strains. This study was conducted by reviewing 131 related articles from Scopus, ISI and PubMed databases. Finally, methods for designing accurate molecular tests to identify disease agents were reported.
Introduction: Acute myeloid leukemia (AML) is a malignant proliferative disease affecting the bone marrow hematopoietic system and has a poor long-term outcome. Exploring genes that affect the malignant proliferation of AML cells can facilitate the accurate diagnosis and treatment of AML. Studies have confirmed that circular RNA (circRNA) is positively correlated with its linear gene expression. Therefore, by exploring the effect of SH3BGRL3 on the malignant proliferation of leukemia, we further studied the role of circRNA produced by its exon cyclization in the occurrence and development of tumors.Methods: Genes with protein-coding function obtained from the TCGA database. we detected the expression of SH3BGRL3 and circRNA_0010984 by real-time quantitative polymerase chain reaction (qRT-PCR). We synthesized plasmid vectors and carried out cell experiments, including cell proliferation, cell cycle and cell differentiation by cell transfection. We also studied the transfection plasmid vector (PLVX-SHRNA2-PURO) combined with a drug (daunorubicin) to observe the therapeutic effect. The miR-375 binding site of circRNA_0010984 was queried using the circinteractome databases, and the relationship was validated by RNA immunoprecipitation and Dual-luciferase reporter assay. Finally, a protein‐protein interaction network was constructed with a STRING database. GO and KEGG functional enrichment identified mRNA-related functions and signaling pathways regulated by miR-375.Results: We identified the related gene SH3BGRL3 in AML and explored the circRNA_0010984 produced by its cyclization. It has a certain effect on the disease progression. In addition, we verified the function of circRNA_0010984. We found that circSH3BGRL3 knockdown specifically inhibited the proliferation of AML cell lines and blocked the cell cycle. We then discussed the related molecular biological mechanisms. CircSH3BGRL3 acts as an endogenous sponge for miR-375 to isolate miR-375 and inhibits its activity, increases the expression of its target YAP1, and ultimately activates the Hippo signaling pathway involved in malignant tumor proliferation.Discussion: We found that SH3BGRL3 and circRNA_0010984 are important to AML. circRNA_0010984 was significantly up-regulated in AML and promoted cell proliferation by regulating miR-375 through molecular sponge action.
Protein aggregation remains a major challenge in the purification of recombinant proteins in both eukaryotic and prokaryotic expression systems. One such protein is the nucleocapsid protein of Crimean Congo Hemorrhagic fever virus (CCHFV), which has high aggregation tendency and rapidly precipitates upon purification by NiNTA chromatography. Using the detergent gradient purification approach reported here, the freshly purified protein by NiNTA chromatography was mixed with the dilution buffer containing a high detergent concentration, followed by overnight freezing at -80 °C. Thawing the resulting mixture at room temperature triggered the formation of a detergent concentration gradient containing the active protein in the low detergent concentration zone towards the top of the gradient. The inactive aggregates migrated to the high detergent concentration zone towards the bottom of the gradient. The method prevented further aggregation and retained the activity of the native protein despite numerous freeze–thaw cycles. This simple approach creates an appropriate microenvironment towards the top of the gradient for correctly folded proteins, and it prevents aggregation by rapidly separating the preformed early aggregates from the correctly folded protein in the mixture. This unique approach will be of potential importance for the biotechnological industry, as well as other fields of protein biochemistry that routinely purify recombinant proteins and face the challenges of protein aggregation.Graphical abstract:
Muhammad Naeem, Saba Manzoor, Mashhud-Ul-Hasan Abid
et al.
With the increasing world population, demand for industrialization has also increased to fulfill humans’ living standards. Fungi are considered a source of essential constituents to produce the biocatalytic enzymes, including amylases, proteases, lipases, and cellulases that contain broad-spectrum industrial and emerging applications. The present review discussed the origin, nature, mechanism of action, emerging aspects of genetic engineering for designing novel proteases, genome editing of fungal strains through CRISPR technology, present challenges and future recommendations of fungal proteases. The emerging evidence revealed that fungal proteases show a protective role to many environmental exposures and discovered that an imbalance of protease inhibitors and proteases in the epithelial barriers leads to the protection of chronic eosinophilic airway inflammation. Moreover, mitoproteases recently were found to execute intense proteolytic processes that are crucial for mitochondrial integrity and homeostasis function, including mitochondrial biogenesis, protein synthesis, and apoptosis. The emerging evidence revealed that CRISPR/Cas9 technology had been successfully developed in various filamentous fungi and higher fungi for editing of specific genes. In addition to medical importance, fungal proteases are extensively used in different industries such as foods to prepare butter, fruits, juices, and cheese, and to increase their shelf life. It is concluded that hydrolysis of proteins in industries is one of the most significant applications of fungal enzymes that led to massive usage of proteomics.
L. M. Polyakov, R. A. Knyasev, M. V. Kotova
et al.
This work shows the ability of apolipoprotein A-I to influence the activity of lysosomal glycosidases in the liver of mice in a model of BCG-induced tuberculous inflammation. The aim of the investigation was to study the activity of lysosomal glycosidases in the liver of mice using a model of BCG-induced tuberculous inflammation after intravenous administration of apolipoprotein A-I. Material and methods. The studies were performed on male CBA mice weighing 20-22 g. Disseminated tuberculous inflammation was modeled by a single intraperitoneal injection of 0.5 mg of BCG vaccine. The activity of lysosomal glycosidases was assessed spectrofluorimetrically by the content of hydrolysis products of the corresponding fluorogenic substrates. Results. Tuberculous inflammation led to a pronounced decrease in the activity of lysosomal glycosidases in the liver. Thus, β-galactosidase was decreased 2.3 times, β-glucosidase – 2.8 times, β-glucuronidase – 2.5 times compared with healthy animals. Intravenous injection of apolipoprotein A-I to animals against the background of BCG infection prevented a significant decrease in the enzymatic activity of glycosidases and these values practically did not differ from the control values. Conclusions. Enzymatic activity of lysosomal glycosidases in the group of mice with intravenous administration of apolipoprotein A-I against the background of BCG infection was 1.5–2 times higher than the corresponding indicators in the group of animals with BCG-infection without administration of apolipoprotein A-I, i.e. without treatment.