Neutrophil migration participates in the side effect of recombinant human tissue plasminogen activator.

AIMS: Ischemic stroke remains a challenge in medical research because of the limited treatment options. Recombinant human tissue plasminogen activator (rtPA) is the primary treatment for recanalization. However, nearly 50% of the patients experience complications that result in ineffective reperfusion. The precise factors contributing to ineffective reperfusion remain unclear; however, recent studies have suggested that immune cells, notably neutrophils, may influence the outcome of rtPA thrombolysis via mechanisms such as the formation of neutrophil extracellular traps. This study aimed to explore the nonthrombolytic effects of rtPA on neutrophils and highlight their contribution to ineffective reperfusion. METHODS: We evaluated the effects of rtPA treatment on middle cerebral artery occlusion in rats. We also assessed neutrophil infiltration and activation after rtPA treatment in vitro and in vivo in a small cohort of patients with massive cerebral ischemia (MCI). RESULTS: rtPA increased neutrophil infiltration into the brain microvessels and worsened blood-brain barrier damage during ischemia. It also increased the neutrophil counts of the patients with MCI. CONCLUSION: Neutrophils play a crucial role in promoting ischemic injury and blood-brain barrier disruption, making them potential therapeutic targets.

Efficiency of recombinant Ybgf in a double antigen-ELISA for the detection of Coxiella antibodies in ruminants.

Q fever is a zoonosis whose main reservoirs are domestic ruminants. Surveillance in these species is carried out mainly with serological tests, which, however, have limited diagnostic performance, and their manufacturing requires laboratories equipped with high biosafety requirements for antigen production. Recombinant ELISAs do not depend on these requirements and, being based on a single antigen, can reduce potential false positivity by identifying antibodies specific to a phase of infection. The aim of this study was to apply a new technology (dual antigen test) to a recombinant protein (Ybgf), an antigen produced in recombinant form and already used in previous studies for the design of an indirect ELISA. The successfully produced recombinant antigen was used to coat 96-well plates and, at the same time, another antigen aliquot was conjugated with HRP to obtain an HRP-conjugated Ybgf. After setting the test conditions, the results obtained with the recombinant double antigen test were compared with those obtained with a commercial assay (considered as reference assay) testing a total of 514 ruminant samples (280 goats and 234 cattle). A concordance of 86.2 and a Cohen's Kappa value of 0.72 were obtained, with no significant difference between the two species tested. Notably, the test proved to be highly specific, having correctly identified 250 out of 253 animals. This research represents an additional effort to use recombinant antigens to enhance serological methods in veterinary medicine. In a "one-health scenario", improving the performance of serological tests used in veterinary practice also means improving the surveillance of this infection.

Superovulating cattle with corifollitropin-alpha, a long-acting recombinant human FSH (rhFSH): Dose-response, half-life, effects on the ovaries, and embryo outcomes.

The aim of this study was to evaluate the superestimulatory and superovulatory responses of cattle treated with corifollitropin-alpha, a long-acting human recombinant FSH (rhFSH). In the first and second experiments, we used Nelore (Bos indicus) heifers previously submitted to follicular wave suppression by active immunization against GnRH. In Experiment 1 (a dose-response study), heifers (n = 20) were randomly allocated into five groups, which received placebo (saline) or a single sc dose of 7.5, 15.0, 22.5 or 30.0 µg rhFSH. The heifers were subjected to daily ovarian scan and blood sampling during 11 days. We observed group, time, and group x time effects (P<0.0001) for both average follicle size and circulating FSH concentrations, with a strong correlation (R = 0.82, P<0.0001) between the area under curve (AUC) for both parameters. The peak concentration of FSH 24h after treatment and average follicle size at all timepoints, however, were similar (P>0.05) between groups 22.5 and 30.0 µg. In Experiment 2, heifers (n = 18) were allocated into three groups, which received (0h) either placebo (control), 25 µg rhFSH or 130 mg pFSH (Folltropin). There was no difference (P>0.05) in average follicle size at any moment, as well as in intrafollicular E2 at 120h or in plasma P4 seven days later between groups rhFSH and pFSH. In Experiment 3, cycling Nelore heifers (n = 20) were subjected to a wave synchronization protocol and superovulated (day 0) using a standard pFSH protocol (120 mg split in eight decreasing im doses) or with a single sc injection of 20 µg rhFSH. The number of follicles >7 mm on day 4 did not differ (P=0.4370). Heifers receiving rhFSH had greater average follicle size on day 4 (P=0.0005), ovulation rate (P<0.0001), and number of CL (P=0.0155), as well as a trend towards a greater number of ova (P=0.07) and viable embryos (P=0.0590). In Experiment 4, superovulation was induced with a single sc injection of 25 µg rhFSH in Girolando and Nelore cows and heifers (n = 20). None of the embryo yield endpoints differed between the two breeds (P>0.05). In conclusion, cattle superstimulation and superovulation can be successfully induced with a single dose of a long-acting rhFSH (corifollitropin-alpha).

Pitfalls of diagnosing pituitary hypoplasia in the patients with short stature.

PURPOSE: Height age (HA) and bone age (BA) delay is well known in the patients with short stature. Therefore assessing pituitary hypoplasia based on chronological age (CA) might cause overdiagnosis of pituitary hypoplasia. We aimed to investigate the diagnostic and prognostic value of the PH and PV based on CA, HA, or BA in the patients with GHD. METHODS: Fifty-seven patients with severe and 40 patients with partial GHD and 39 patients with ISS assigned to the study. For defining the most accurate diagnosis of pituitary hypoplasia, PH and PV were evaluated based on CA, BA and HA. The relationship of each method with clinical features was examined. RESULTS: The mean PV was significantly larger in patients with ISS compared to the GH-deficient patients. PV was more correlated with clinical features including height SDS, stimulated GH concentration, IGF-1 and IGFBP-3 SDS, height velocity before and after rGH therapy. We found BA-based PV could discriminate GHD from ISS (Sensitivity: 17%, specificity: 98%, positive predictive value: 94%, negative predictive value: 39%), compared to the other methods based on PH or PV respect to CA and HA. 3% of patients with ISS, 17% of patients with GHD had pituitary hypoplasia based on PV-BA. CONCLUSION: PV based on BA, has the most accurate diagnostic value for defining pituitary hypoplasia. But it should be kept in mind that there might be still misdiagnosed patients by this method. PV is also a significant predictor for the rGH response.

Optimization of extended Kozak elements enhances recombinant proteins expression in CHO cells.

In eukaryotes, the localization of small ribosomal subunits to mRNA transcripts requires the translation of Kozak elements at the starting site. The sequence of Kozak elements affects the translation efficiency of protein synthesis. However, whether the upstream nucleotide of Kozak sequence affects the expression of recombinant proteins in Chinese hamster ovary (CHO) cells remains unclear. In order to find the optimal sequence to enhance recombinant proteins expression in CHO cells, -10 to +4 sequences around ATG in 100 CHO genes were compared, and the extended Kozak elements with different translation intensities were constructed. Using the classic Kozak element as control, the effects of optimized extended Kozak elements on the secreted alkaline phosphatase (SEAP) and human serum albumin (HSA) gene were studied. The results showed that the optimized extended Kozak sequence can enhance the stable expression level of recombinant proteins in CHO cells. Furthermore, it was found that the increased expression level of the recombinant protein was not related with higher transcription level. In summary, optimizing extended Kozak elements can enhance the expression of recombinant proteins in CHO cells, which contributes to the construction of an efficient expression system for CHO cells.

Construction of Recombinant Baculoviruses.

The success of using the insect cell-baculovirus expression technology (BEST) relies on the efficient construction of recombinant baculovirus with genetic stability and high productivity, ideally within a short time period. Generation of recombinant baculoviruses requires the transfection of insect cells, harvesting of recombinant baculovirus pools, isolation of plaques, and the expansion of baculovirus stocks for their use for recombinant protein production. Moreover, many options exist for selecting the genetic elements to be present in the recombinant baculovirus. This chapter describes the most commonly used homologous recombination systems for the production of recombinant baculoviruses, as well as strategies to maximize generation efficiency and recombinant protein or baculovirus production. The key steps for generating baculovirus stocks and troubleshooting strategies are described.

Recombinant AAV Production.

The insect cell-baculovirus expression vector (IC-BEV) platform has enabled small research-scale and large commercial-scale production of recombinant proteins and therapeutic biologics including recombinant adeno-associated virus (rAAV)-based gene delivery vectors. The wide use of this platform is comparable with other mammalian cell line-based platforms due to its simplicity, high-yield, comparable quality attributes, and robust bioprocessing features. In this chapter, we describe a rAAV production protocol employing one of the recent modifications of the One-Bac platform that consists of a stable transformed Sf9 cell line carrying AAV Rep2/Cap5 genes that are induced upon infection with a single recombinant baculovirus expression vector harboring the transgene of interest (rAAV genome). The overall protocol consists of essential steps including rBEV working stock preparation, rAAV production, and centrifugation-based clarification of cell culture lysate. The same protocol can also be applied for rAAV vector production using traditional Three-Bac, Two-Bac, and Mono-Bac platforms without requiring significant changes.

Recombinant AAV Purification.

Purification of rAAV is a crucial unit operation of the AAV production process. It enables the capture of AAV and removal of contaminants such as host cell proteins, host cell DNA, and other cell culture-related impurities. Here we describe the purification of rAAV produced in insect cells Sf9/rBEV by immuno-affinity capture chromatography. The method is fully scale-amenable unlike other traditional purification methods based on ultracentrifugation. The method reported herein has two main steps: (1) the clarification of cell lysate by depth filtration and (2) the selective capture and single-step purification of AAV via immune-affinity chromatography. This purification method has been successfully implemented to purify the majority of wild-type AAV serotypes.

Nonviral Platform for Expression of Recombinant Protein in Insect Cells.

Nonviral transfection has been used to express various recombinant proteins, therapeutics, and virus-like particles (VLP) in mammalian and insect cells. Virus-free methods for protein expression require fewer steps for obtaining protein expression by eliminating virus amplification and measuring the infectivity of the virus. The nonviral method uses a nonlytic plasmid to transfect the gene of interest into the insect cells instead of using baculovirus, a lytic system. In this chapter, we describe one of the transfection methods, which uses polyethyleneimine (PEI) as a DNA delivery material into the insect cells to express the recombinant protein in both adherent and suspension cells.

Functional Baculovirus Particle Quantification via Plaque Assay.

Plaque assay method enables the quantification of infectious baculovirus when defined as plaque forming units (PFU). It allows to determine the amount of infectious virus needed to infect the cells at a specific multiplicity of infection (MOI). Serial dilutions of baculovirus stock are added to the Sf9 cells monolayer followed by addition of 5% Agarose overlay. Six days after infection clear infection halos are observed using a neutral red solution. Here we describe the quantification of recombinant baculovirus expression vector (rBEV) carrying a transgene in an rAAV expression cassette. Reproducible quantification of PFU is obtained with this method.

Genome-Wide CRISPRi Screening of Key Genes for Recombinant Protein Expression in Bacillus Subtilis.

Bacillus subtilis is an industrially important microorganism that is often used as a microbial cell factory for the production of recombinant proteins due to its food safety, rapid growth, and powerful secretory capacity. However, the lack of data on functional genes related to recombinant protein production has hindered the further development of B. subtilis cell factories. Here, a strategy combining genome-wide CRISPRi screening and targeted CRISPRa activation to enhance recombinant protein expression is proposed. First, a CRISPRi library covering a total of 4225 coding genes (99.7%) in the B. subtilis genome and built the corresponding high-throughput screening methods is constructed. Twelve key genes for recombinant protein expression are identified, including targets without relevant functional annotations. Meanwhile, the transcription of recombinant protein genes by CRISPRa is up-regulated. These screened or selected genes can be easily applied to metabolic engineering by constructing sgRNA arrays. The relationship between differential pathways and recombinant protein expression in engineered strains by transcriptome analysis is also revealed. High-density fermentation and generalisability validation results prove the reliability of the strategy. This method can be extended to other industrial hosts to support functional gene annotation and the design of novel cell factories.

Efficacy of recombinant human epidermal growth factor plus sodium hyaluronate eye drops in diabetic dry eye post-cataract surgery.

BACKGROUND: Dry eye syndrome (DES) after diabetic cataract surgery can seriously affect the patient's quality of life. Therefore, effective alleviation of symptoms in patients with this disease has important clinical significance. AIM: To explore the clinical effect of recombinant human epidermal growth factor (rhEGF) plus sodium hyaluronate (SH) eye drops on DES after cataract surgery in patients with diabetes. METHODS: We retrospectively evaluated 82 patients with diabetes who experienced DES after cataract surgery at Tianjin Beichen Hospital, Affiliated Hospital of Nankai University between April 2021 and April 2023. They were classified into an observation group (42 cases, rhEGF + SH eye drops) and a control group (40 cases, SH eye drops alone), depending on the different treatment schemes. The thera-peutic efficacy, dry eye symptom score, tear film breakup time (TFBUT), basic tear secretion score [assessed using Schirmer I test (SIt)], corneal fluorescein staining (FL) score, tear inflammatory markers, adverse reactions during treatment, and treatment satisfaction were compared between the two groups. RESULTS: Therapeutic efficacy was higher in the observation group compared with the control group. Both groups showed improved TFBUT and dry eye, as well as improved SIt and FL scores after treatment, with a more pronounced improvement in the observation group. Although no marked differences in adverse reactions were observed between the two groups, treatment satisfaction was higher in the observation group. CONCLUSION: rhEGF + SH eye drops rendered clinical benefits to patients by effectively ameliorating dry eye and visual impairment with favorable efficacy, fewer adverse reactions, and high safety levels. Thus, this treatment should be promoted in clinical practice.

Harnessing the potential of microalgae for the production of monoclonal antibodies and other recombinant proteins.

Monoclonal antibodies (mAbs) have become indispensable tools in various fields, from research to therapeutics, diagnostics, and industries. However, their production, primarily in mammalian cell culture systems, is cost-intensive and resource-demanding. Microalgae, diverse photosynthetic microorganisms, are gaining attention as a favorable option for manufacturing mAbs and various other recombinant proteins. This review explores the potential of microalgae as a robust expression system for biomanufacturing high-value proteins. It also highlights the diversity of microalgae species suitable for recombinant protein. Nuclear and chloroplast genomes of some microalgae have been engineered to express mAbs and other valuable proteins. Codon optimization, vector construction, and other genetic engineering techniques have significantly improved recombinant protein expression in microalgae. These accomplishments demonstrate the potential of microalgae for biopharmaceutical manufacturing. Microalgal biotechnology holds promise for revolutionizing the production of mAbs and other therapeutic proteins, offering a sustainable and cost-effective solution to address critical healthcare needs.

Cost-effectiveness of recombinant influenza vaccine compared with standard dose influenza vaccine in adults 18-64 years of age.

BACKGROUND: The Advisory Committee on Immunization Practices (ACIP) uses the Evidence to Recommendations Framework that includes cost-effectiveness analyses (CEA) for determining vaccine recommendations. ACIP's preference for protecting adults ≥ 65 years is enhanced vaccines, including recombinant influenza vaccine (RIV4), adjuvanted or high dose influenza vaccine. Less is known about the CEA of enhanced vaccines for younger adults. METHODS: We used decision analysis modeling from a societal perspective to determine the cost-effectiveness, measured in quality adjusted life years (QALYs), of RIV4 compared with standard dose quadrivalent influenza vaccine (SD-IIV4) in adults 18-64 years old. Model inputs included 2018-2020 vaccine effectiveness (VE) estimates based on medical record data from a large local health system, 2019-2020 national vaccination and influenza epidemic parameters, with costs and population distributions fitted to the season. RESULTS: Among adults ages 18-64 years, RIV4 cost $94,186/QALY gained, compared to SD-IIV4. Among those 50-64 years old, RIV4 was relatively more cost-effective ($61,329/QALY gained). Cost-effectiveness estimates for 18-64-year-olds were sensitive to the absolute difference in VE between SD-IIV4 and RIV4, among other parameters. Use of RIV4 in 18-64-year-olds would result in fewer cases (669,984), outpatient visits (261,293), hospitalizations (20,046) and deaths (1,018) annually. The majority (59 %; 597 of 1018) of the decreases in deaths occurred in the 50-64-year-olds. CONCLUSIONS: While RIV4 was effective and cost-effective relative to SD-IIV4 for both 50-64-year-old and 18-64-year-old adults, cost-effectiveness was sensitive to small changes in parameters among 18-64-year-olds. Because substantial public health benefits occur with enhanced vaccines, health systems and policy makers may opt for preferential product use in select age/risk groups (e.g., 50-64 year olds) to maximize their cost-benefit ratios.

Interferon alpha-2b treatment for exophytic nasal papillomas and human papillomavirus infection.

OBJECTIVES: Exophytic Sinonasal Papilloma (ESP) is a benign tumor of the sinonasal tract. Complete surgical excision by endoscopic surgery is the treatment of choice. However, a high recurrence rate (36% at 5-year follow-up) is associated with this method, which may indicate the presence of microorganisms such as Human Papillomavirus (HPV). It is important to note that the standard treatment for ESP does not include antiviral drugs. In our study, we are testing the effectiveness of an interferon-containing drug in reducing recurrence and postoperative reactions in patients with ESP. METHODS: We included 78 patients aged 23-83 years with a confirmed diagnosis of ESP by rhinoscopy and nasal endoscopy and a positive PCR test for HPV in nasal scrapings. To compare the results, we divided the patients into main and control groups. The main group received recombinant human interferon after surgery, while the control group did not receive the drug. We performed a statistical analysis to compare the proportion of patients without reactive manifestations at different stages of the postoperative period, as well as to compare the proportion of patients with recurrent ESP at certain stages of observation. RESULTS: The introduction of recombinant human interferon accelerated the resolution of postoperative reactions and promoted the healing of the nasal mucosa after surgical removal of the ESP. We also found a statistically significant association between treatment with recombinant interferon and a reduction in the recurrence rate of ESP. CONCLUSION: According to the results of the study, it was found that in the main group of patients who received rhIFN-α2b (recombinant human Interferon alpha 2b) in the postoperative period, the frequency of relapses of ESP and the time of postoperative recovery were significantly lower than in patients in the control group who did not take the drug. LEVEL OF EVIDENCE: Cohort Study.

Postpartum Hemorrhage: Enhancing Outcomes for Mothers by Effective Management.

Postpartum hemorrhage (PPH) remains a significant contributor to maternal morbidity and mortality worldwide. In India, PPH affects approximately 12% of women. The prevention and management of PPH are the significant challenges in obstetrics, with accurate assessment of blood loss and timely intervention being critical. Active Management of the Third Stage of Labor is a gold standard strategy for prevention. Recent advancements in PPH management include the use of recombinant activated factor VIIa, which has shown promise in decreasing the need for invasive procedures and second-line therapies. Additionally, surgical and radiological interventions have been effective in cases of refractory PPH. Overall, ongoing research and advancements in PPH management continue to enhance the quality of care and maternal outcomes experiencing this potentially life-threatening complication of childbirth. This editorial explores prevention and management of Atonic PPH with encompassing medical and surgical strategies, to enhance understanding and optimize clinical care for mothers at risk of this obstetric emergency.

Paradoxical cell targeting of calreticulin-empowered, protein-only nanoparticles.

Surface-exposed calreticulin (CRT) serves as a crucial cell damage-associated molecular pattern for immunogenic apoptosis, by generating an "eat me" signal to macrophages. Aiming at precision immunotherapies we intended to artificially label tumoral cells in vivo with a recombinant CRT, in a targeted way. For that, we have constructed a CRT fusion protein intended to surface attach CXCR4+ cancer cells, to stimulate their immunological destruction. As a targeting ligand of the CRT construct and to drive its specific cell adhesion, we used the peptide V1, a derivative of the vMIP-II cytokine and an antagonist of CXCR4. The modular protein tends to self-assemble as regular 16 nm nanoparticles, assisted by ionic Zn. Through both in vivo and in vitro experiments, we have determined that CRT itself confers cell targeting capabilities to the construct overcoming those of V1, that are only moderate. In particular, CRT binds HeLa cells in absence of further internalization, by a route fully independent of CXCR4. Furthermore, by cytometry in THP-1 cells, we observed that the binding of the protein is preferential for dead cells over live cells, a fact that cannot be associated to a mere artefactual adsorption. These data are discussed in the context of the oligomerizing properties of CRT and the potential clinical applicability of proteins and protein materials functionalized with this novel cell surface ligand.

Mass production and characterization of an endoglucanase from Coleoptera insect (Monochamus saltuarius) in yeast Kluyveromyceslactis.

To harness the diverse industrial applications of cellulase, including its use in the food, pulp, textile, agriculture, and biofuel sectors, this study focused on the high-yield production of a bioactive insect-derived endoglucanase, Monochamus saltuarius glycoside hydrolase family 5 (MsGHF5). MsGHF5 was introduced into the genome of Kluyveromyces lactis to maintain expression stability, and mass production of the enzyme was induced using fed-batch fermentation. After 40 h of cultivation, recombinant MsGHF5 was successfully produced in the culture broth, with a yield of 29,000 U/L, upon galactose induction. The optimal conditions for the activity of purified MsGHF5 were determined to be a pH of 5 and a temperature of 35 °C, with the presence of ferrous ions enhancing the enzymatic activity by up to 1.5-fold. Notably, the activity of MsGHF5 produced in K. lactis was significantly higher than that produced in Escherichia coli, suggesting that glycosylation is crucial for the functional performance of the enzyme. This study highlights the potential use of K. lactis as a host for the production of bioactive MsGHF5, thus paving the way for its application in various industrial sectors.

Sequence Notes: Near Full-Length Genome Analysis of the First-Reported HIV-1 Circulating Recombinant Form (CRF)_10CD in Uganda.

HIV-1 is characterized by remarkable genetic diversity resulting from its high replication rate, error-prone reverse transcriptase enzyme and recombination events. In Uganda, HIV-1 subtype diversity is mostly dominated by subtypes A, D, and A1/D Unique Recombinant Forms (URFs). In this study, deep sequences of HIV from patients with known antiretroviral therapy (ART) status were analyzed to determine the subtypes and to identify drug-resistance mutations circulating in the study population. Of the 187 participant samples processed for next-generation sequencing (NGS), 137 (73%) were successfully classified. The majority of HIV-1 strains were classified as subtype A (75, 55%), D (43, 31%), with other subtypes including C (3, 2%), A1/D (9, 7%) and CRF10_CD (1, <1%). Recombinant analysis of nine complete A1/D HIV genomes identified novel recombination patterns described herein. Furthermore, we report for the first time in Uganda, an HIV-1 CRF10_CD strain from a fisherfolk in a Lake Victoria Island fishing community.

Effects of Lipoproteins on Metabolic Health.

Lipids are primarily transported in the bloodstream by lipoproteins, which are macromolecules of lipids and conjugated proteins also known as apolipoproteins. The processes of lipoprotein assembly, secretion, transportation, modification, and clearance are crucial components of maintaining a healthy lipid metabolism. Disruption in any of these steps results in pathophysiological abnormalities such as dyslipidemia, obesity, insulin resistance, inflammation, atherosclerosis, peripheral artery disease, and cardiovascular diseases. By studying these genetic mutations, researchers can gain valuable insights into the underlying mechanisms that govern the relationship between protein structure and its physiological role. These lipoproteins, including HDL, LDL, lipoprotein(a), and VLDL, mainly serve the purpose of transporting lipids between tissues and organs. However, studies have provided evidence that apo(a) also possesses protective properties against pathogens. In the future, the field of study will be significantly influenced by the integration of recombinant DNA technology and human site-specific mutagenesis for treating hereditary disorders. Several medications are available for the treatment of dyslipoproteinemia. These include statins, fibrates, ezetimibe, niacin, PCSK9 inhibitors, evinacumab, DPP 4 inhibitors, glucagon-like peptide-1 receptor agonists GLP1RAs, GLP-1, and GIP dual receptor agonists, in addition to SGLT2 inhibitors. This current review article exhibits, for the first time, a comprehensive reflection of the available body of publications concerning the impact of lipoproteins on metabolic well-being across various pathological states.