Production of potent long-lasting consensus interferon using albumin fusion technology in Pichia pastoris expression system.

Consensus interferon (cIFN) is a wholly synthetic therapeutic protein which is used to treat hepatitis C/B and certain types of malignancies. It has short serum half-life, therefore, to maintain its therapeutic level in the human body it requires thrice-weekly administration. Various strategies like PEGylation and micro-encapsulation have been developed during the last few years to enhance the pharmacokinetics of small therapeutic peptides. This study executed the human albumin-fusion technology, a simple and flexible approach to extend the serum circulating half-life of cIFN, because human serum albumin (HSA) has long circulating half-life (19 days) and very minute immunological activities. We integrated the codon-optimized HSA-cIFN fusion gene into Pichia pastoris genome by homologous recombination. The selection of hyper-resistant P. pastoris clone against Zeocin™ achieved a high-level secretory expression (250 mg/L) of fusion protein. HSA-cIFN fusion protein was purified using one-step purification by affinity chromatography with 34% recovery. The SDS-PAGE and SEC-HPLC analysis confirmed the final purified product has molecular weight of 87 kDa with 98% purity. Western blot analysis using anti-IFN antibodies further verified the purified HSA-cIFN fusion protein. The specific biological activity was 2.1 × 106 IU/mg as assessed by cytopathic inhibition assay, and half-life of fusion protein was estimated by in vitro thermal and proteolytic stability studies. This work concludes that by using albumin fusion technology, codon optimization and one-step purification a high yield of 86 mg/L of biologically active protein with improved serum half-life was obtained.

Evaluating the ion exchange chromatography for matrix-assisted PEGylation and purification of consensus interferon.

Scientists have implemented protein-PEGylation technology for boosting-up the pharmacokinetics and stability of recombinant therapeutic proteins. In the present study, (a) matrix-assisted PEGylation was compared with solution-phase PEGylation and (b) matrix-assisted PEGylation was performed with different ion exchange resins for impact of chromatography medium on yield and purity of PEGylated product. DEAE Sepharose CL 6B, DEAE Fracto gel, and Macro cap Q ion exchange chromatography medium were compared for on column PEGylation and purification of cIFN. A MSC-PEG of 12.0 KDa was selected. cIFN was bound to ion exchange medium, and PEG solution was passed through resin for 180 Min, and protein was eluted by sodium chloride linear gradient. Yield and purity for mono-PEGylated cIFN with Macro cap Q matrix was 75% and 99%, respectively, whereas for DEAE Sepharose was 45% and 60%. DEAE Fracto gelTM purity was 85% with 50% yield of mono-PEGylated cIFN. Further investigation of in vitro biological activities demonstrated that about 30% antiviral activity was reduced as compared to unmodified cIFN. However, thermal stability was significantly improved. The present study proved that matrix-assisted PEGylation can improve the yield and purity of mono-PEGylated product, and Macro Cap resin provided the highest yield of a homogeneous product. In present study, (a) matrix-assisted PEGylation was compared with solution-phase PEGylation and (b) matrix-assisted PEGylation was performed with different ion exchange resins for impact of chromatography medium on yield and purity of PEGylated product. Matrix-assisted PEGylation increases the yield of mono-PEGylated product and further Macro CapTM produced highest yield and purity of PEGylated cIFN.

Enhancing recombinant interleukin-6 production yield by fermentation optimization, two-step denaturing, and one-step purification.

Interleukin-6 a pleiotropic cytokine involved in a wide range of biological activities. So the large-scale production of biologically active recombinant human interleukin-6 is important for its structural and functional studies. Here, we report an optimized method for shake flask fermentation and a simplified high-yield purification procedure for the recombinant interleukin-6. This high-yield expression method not only involves the optimization of the fermentation condition but also the single step purification method as well as a two-step denaturing and one-step refolding process. This approach replaces the more conventional procedure of protein solubilization and refolding. Through applying these strategies, the final cell density and overall product yield of the recombinant human interleukin-6 were obtained as 20.4 g as cell biomass and 150 mg as purified active protein from the I-L of the culture. The purified protein was characterized by HPLC and SDS-PAGE. The results of the current work demonstrate that the described method may be used to develop the process for industrial-scale production of the biologically active recombinant interleukin-6 protein.

Adult Influenza A (H1N1) Related Encephalitis: A Case Report.

The year 2009-2010 saw H1N1 influenza outbreaks occurring in almost all countries of the world, causing the WHO to declare it a pandemic of an alert level of 6. In India, H1N1 influenza outbreaks were again reported in late 2014 and early 2015. Since then, sporadic cases of H1N1 influenza have been reported. H1N1 influenza usually presents itself with respiratory tract symptoms. In a minority of patients, abdominal symptoms may occur as well. Acute influenza-associated encephalopathy/encephalitis mostly occurs in the pediatric population, whereas in adults, it is a rare complication. The incidence of neurological complications appears to have increased after the 2009 H1N1 influenza A virus pandemic. We would like to draw attention to an adult patient case who initially presented with respiratory symptoms but then deteriorated and developed encephalitis, which is rarely reported. As per literature reviewed by Victoria Bangualid and Judith Berger on PubMed, only 21 cases of neurological complications were found in adult influenza A patients, out of whom 8 had encephalopathy.

Evaluating the autoinduction expression system and one-step purification for high-level expression and purification of gallbladder-derived rhIL-1Ra.

Recent advancement in fermentation technologies resulted in the increased yields of recombinant proteins of biopharmaceutical and medicinal importance. Consequently, there is an important task to develop simple and easily scalable methods that can facilitate the production of high-quality recombinant protein. Most of the recent reports described the expression of recombinant human IL-1 receptor antagonist (rhIL-1Ra) in Escherichia coli using isopropyl-ß-d-thiogalacto pyranoside (IPTG), a nonmetabolizable and expensive compound, as an expression inducer. In this study, we describe the expression and one-step purification of gallbladder-derived rhIL-1Ra by autoinduction in E. coli. This method includes special media that automatically induce the target protein expression from T7 promoter and allow the production of the target protein in high yield than the conventional IPTG induction method. In addition to fermentation process improvements, one-step purification strategy is essential to make the process economical. We developed a single-step cation exchange chromatography and obtained 300 mg/L of rhIL-1Ra with 98% purity. Purified protein was characterized by SDS-PAGE and Ion exchange HPLC (IEX-HPLC). The described method can be used to scale up the production of rhIL-1Ra and other recombinant proteins.

Bioactivity studies of Huh-7 cells derived human epidermal growth factor expressed in Pichia pastoris.

Previously, we have reported cloning of human epidermal growth factor gene from Huh-7 cells and its extracellular expression in Pichia pastoris. The presented work is a detailed report regarding molecular characterization of Huh-7 cells-derived hEGF expressed in Pichia pastoris with special reference to its glycosylation profiling and bioactivity studies. Densitometric scanning of SDS-PAGE separated extracellular proteins from hEGF recombinant Pichia pastoris strain indicated that about 84% of the extracellular proteins were glycosylated. Size exclusion chromatography using Superdex 75 prep grade column was successfully utilized to separate fractions containing glycosylated and non-glycosylated extracellular proteins. In dot blot assay, hEGF was detected in both glycosylated and non-glycosylated fractions. Bioactivity assays revealed that both glycosylated and non-glycosylated fractions were bioactive as determined by cell viability assay. It was also observed that hEGF present in non-glycosylated fraction was relatively more bioactive than hEGF present in glycosylated fraction.

Simple procedure applying lactose induction and one-step purification for high-yield production of rhCIFN.

Recombinant consensus interferon (CIFN) is a therapeutic protein with molecular weight of 19.5 kDa having broad spectrum antiviral activity. Recombinant human CIFN (rhCIFN) has previously been expressed in Escherichia coli using isopropyl-ß-d-thiogalactopyranoside (IPTG), a non-metabolizable and expensive compound, as inducer. For economical and commercial-scale recombinant protein production, it is greatly needed to increase the product yield in a limited time frame to reduce the processing cost. To reduce the cost of production of rhCIFN in E. coli, induction was accomplished by using lactose instead of IPTG. Lactose induction (14 g/L) in shake flask experiment resulted in higher yield as compared with 1 mM IPTG. Finally, with single-step purification on DEAE sepharose, 150 mg/L of >98% pure rhCIFN was achieved. In the present study, an attempt was made to develop a low cost process for producing quality product with high purity. Methods devised may be helpful for pilot-scale production of recombinant proteins at low cost.

Optimization of conditions for high-level expression and purification of human recombinant consensus interferon (rh-cIFN) and its characterization.

Recombinant human consensus interferon (rh-cIFN) is an artificially engineered interferon (IFN) developed by recombining and reordering the protein sequences that exist in standard IFN. This recombination resulted into a drug that has the potential to work better than natural, standard IFN. In this study, we described optimized conditions for high-level expression and recovery of biologically active consensus IFN from inclusion bodies (IBs). A synthetic gene coding 166 amino acids of consensus IFN was cloned under the T7 promoter. Escherichia coli strain BL21DE3Plys was used to transform expression construct. For high-level expression, shake-flask fermentation conditions were standardized. For isolation of IBs, the sonication method was optimized. A variety of chaotropic agents including guanidine hydrochloride, urea, SDS, and detergents were studied for solubilization of IBs. For renaturation of solubilized denatured protein by the dilution process, parameters of dilution factor, temperature, and l-arginine were optimized. A one-step chromatography method was developed for high-yield purification of consensus IFN. rh-cIFN was characterized by SDS-PAGE, Western blot, and high-performance liquid chromatography. Purified protein has a molecular weight of 19.5 kDa and specific activity was 2.0 × 10(8) as determined by the cytopathic inhibition assay. This study concludes that by using optimized conditions, we obtained a yield of 100 mg/L of biologically active rh-cIFN, which is highest ever reported according to available data.

Studies to analyse the relationship between IFNα2b gene dosage and its expression, using a Pichia pastoris-based expression system.

Human interferon α2b (hIFNα2b) is the most important member of the interferon family. Escherichia coli, yeasts, mammalian cell cultures and baculovirus-infected insect cells have been used for expressing recombinant human interferon. Recently a Pichia pastoris-based expression system has emerged as an attractive system for producing functional human recombinant IFNα2b. In this regard, gene dosage is considered an important factor in obtaining the optimum expression of recombinant protein, which may vary from one protein to another. In the present study we have shown the effect of IFNα2b gene dosage on extracellular expression of IFNα2b recombinant protein from P. pastoris. Constructs containing from one to five repeats of IFNα2b-expressing cassettes were created via an in vitro multimerization approach. P. pastoris host strain X-33 was transformed using these expression cassettes. Groups of P. pastoris clones transformed with different copies of the IFNα2b expression cassette were screened for intrachromosomal integration. The IFNα2b expression level of stable transformants was checked. The copy number of integrated IFNα2b was determined by performing qPCR of genomic DNA of recombinant P. patoris clones. It was observed that an increase in copy number generally had a positive effect on the expression level of IFNα2b protein. Regarding the performance of multicopy strains, those obtained from transformation of multicopy vectors showed relatively high expression, compared to those generated using transformation vector having only one copy of IFNα2b. It was also observed that an increase in drug resistance of a clone did not guarantee its high expression, as integration of a marker gene did not always correlate with integration of the gene of interest.

Expression line approach to recombinant human epidermal growth factor into the yeast, Pichia pastoris from Huh-7 cell line.

Beta-urogastrone also known as human epidermal growth factor is a key member of epidermal growth factor family having role in cell proliferation and differentiation in vivo as well as in vitro. Human epidermal growth factor gene has been isolated from different tissues but the method of isolation is technically difficult and complicated as it deals with biopsies. Here we isolated mature partial human epidermal growth factor gene from Huh-7 cell line, amplified and abridged toward mature coding region with three steps PCR, sequenced for homology with wild type human epidermal growth factor gene, inbuilt with sites of interest and cloned in Pichia pastoris for expression study. Isolated mature human epidermal growth factor gene from Huh-7 cell line showed 100 % sequence homology to wild type human epidermal growth factor gene and gives the native expression for human epidermal growth factor peptide. In this study we report that Huh-7 cell line is an easy source for the particular gene of human epidermal growth factor isolation and we are also suggesting P. pastoris is an expression system to produce recombinant human epidermal growth factor of the therapeutic importance resembling to the natural human system.

Matrix-assisted refolding and purification of placenta-derived recombinant human interleukin-6 produced in Escherichia coli.

Biological activity of human interleukin-6 (IL-6) is associated with a vast number of diseases such as rheumatoid arthritis, sepsis, and severe inflammatory diseases. In this study, human IL-6 cDNA was isolated from a cDNA library that was constructed with mRNA derived from human placental tissues. Subsequently, the complete human IL-6 cDNA was cloned and expressed in BL21DE3 cells. The recombinant human IL-6 (rhIL-6) protein was expressed in a form of an insoluble inclusion body. Inclusion bodies were solubilized under denaturing conditions and purified by immobilized metal affinity chromatography with gradual on-column refolding by the gradient elution method (from 6 to 0 M urea). The protein was purified to apparent homogeneity of about 99% with a yield of 50 mg/L. The purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion high-performance liquid chromatography, and Western blotting analysis. The bioactivity was assessed by proliferation assay of TF-1 cells in a dose-dependent manner. The present study confirms the expression of the placenta-derived IL-6 gene in a prokaryotic expression system and matrix-assisted on-column refolding and purification of rhIL-6 by immobilized metal affinity chromatography.

Deciphering mycobiota and its functional dynamics in root hairs of Rhododendron campanulatum D. Don through Next-gen sequencing.

The Himalayas provide unique opportunities for the extension of shrubs beyond the upper limit of the tree. However, little is known about the limitation of the biotic factors belowground of shrub growth at these cruising altitudes. To fill this gap, the present study deals with the documentation of root-associated microbiota with their predicted functional profiles and interactions in the host Rhododendron campanulatum, a krummholz species. While processing 12 root samples of R. campanulatum from the sites using Omics we could identify 134 root-associated fungal species belonging to 104 genera, 74 families, 39 orders, 17 classes, and 5 phyla. The root-associated microbiota members of Ascomycota were unambiguously dominant followed by Basidiomycota. Using FUNGuild, we reported that symbiotroph and pathotroph as abundant trophic modes. Furthermore, FUNGuild revealed the dominant prevalence of the saptroptroph guild followed by plant pathogens and wood saprotrophs. Alpha diversity was significantly different at the sites. The heatmap dendrogram showed the correlation between various soil nutrients and some fungal species. The study paves the way for a more in-depth exploration of unidentified root fungal symbionts, their interactions and their probable functional roles, which may serve as an important factor for the growth and conservation of these high-altitude ericaceous plants.

A mutation in SLC24A1 implicated in autosomal-recessive congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder that can be associated with impaired night vision. The last decade has witnessed huge progress in ophthalmic genetics, including the identification of three genes implicated in the pathogenicity of autosomal-recessive CSNB. However, not all patients studied could be associated with mutations in these genes and thus other genes certainly underlie this disorder. Here, we report a large multigeneration family with five affected individuals manifesting symptoms of night blindness. A genome-wide scan localized the disease interval to chromosome 15q, and recombination events in affected individuals refined the critical interval to a 10.41 cM (6.53 Mb) region that harbors SLC24A1, a member of the solute carrier protein superfamily. Sequencing of all the coding exons identified a 2 bp deletion in exon 2: c.1613_1614del, which is predicted to result in a frame shift that leads to premature termination of SLC24A1 (p.F538CfsX23) and segregates with the disorder under an autosomal-recessive model. Expression analysis using mouse ocular tissues shows that Slc24a1 is expressed in the retina around postnatal day 7. In situ and immunohistological studies localized both SLC24A1 and Slc24a1 to the inner segment, outer and inner nuclear layers, and ganglion cells of the retina, respectively. Our data expand the genetic basis of CSNB and highlight the indispensible function of SLC24A1 in retinal function and/or maintenance in humans.

Obstructive sleep apnoea in adults.

Obstructive sleep apnoea (OSA) is characterised by repetitive closure of the upper airway, repetitive oxygen desaturations and sleep fragmentation. The prevalence of adult OSA is increasing because of a worldwide increase in obesity and the ageing of populations. OSA presents with a variety of symptoms the most prominent of which are snoring and daytime tiredness. Interestingly though, a significant proportion of OSA sufferers report little or no daytime symptoms. OSA has been associated with an increased risk of cardiovascular disease, cognitive abnormalities and mental health problems. Randomised controlled trial evidence is awaited to confirm a causal relationship between OSA and these various disorders. The gold standard diagnostic investigation for OSA is overnight laboratory-based polysomnography (sleep study), however, ambulatory models of care incorporating screening questionnaires and home sleep studies have been recently evaluated and are now being incorporated into routine clinical practice. Patients with OSA are very often obese and exhibit a range of comorbidities, such as hypertension, depression and diabetes. Management, therefore, needs to be based on a multidisciplinary and holistic approach which includes lifestyle modifications. Continuous positive airway pressure (CPAP) is the first-line therapy for severe OSA. Oral appliances should be considered in patients with mild or moderate disease, or in those unable to tolerate CPAP. New, minimally invasive surgical techniques are currently being developed to achieve better patient outcomes and reduce surgical morbidity. Successful long-term management of OSA requires careful patient education, enlistment of the family's support and the adoption of self-management and patient goal-setting principles.

PEG-interferon conjugates: effects of length and structure of linker.

Di-branched (Y-shaped) polyethylene glycols (PEGs) are considered more effective than linear molecules to enhance the efficacy of the conjugated drug. In the present study interferon α-2a was conjugated with three different 40 KDa di-branched PEGs. The results of this study show that length and/or the structure of linker between PEG and the protein is also involved in the synthesis, in vitro biological activity and stability of the conjugate. Three conjugates i.e., mPEG2L-IFN, mPEG2P-IFN and mPEG(2)M-IFN yielded 25%, 24% and 17%, with bioactivities 2.8 x 10(6) IU/mg, 3.95 x 10(6) IU/mg and 6.7 x 10(6) IU/mg, respectively. The order of bioactivity stability is mPEG2L-IFN > mPEG2P-IFN > mPEG2M-IFN > IFN (native). We report that although lengthy linkers are more reactive in terms of conjugation, they have opposite effect on the in vitro bioactivity of the conjugate. PEGylation as a whole increases the stability of the conjugate, and linkers also add in stability.

Role of robotic approach in ileal pouch-anal anastomosis (IPAA): A systematic review of the literature.

Restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) has become standard surgical treatment of choice in patients with ulcerative colitis (UC) and familial adenomatous polyposis (FAP) in which the medical management fails. Despite the wide use of laparoscopic method, the enhanced and innovative features that come with the robotic platform, such as endo-wrist technology, 3D visualization, surgeon-controlled camera and motion scaling, make it an appealing choice. This study aims to investigate the feasibility and safety of robotic approach for proctectomy or proctocolectomy with IPAA as compared to conventional laparoscopic approach. A systematic review was completed for studies done between 2010 and 2022 comparing the robotic approach with the laparoscopic approach. Nine studies were found to be feasible to be included in this review. In terms of the outcomes, although the mean operating time was slightly higher than the laparoscopic approach, the other outcomes, such as mean blood loss, return of the bowel movement, mean hospital stay, and conversion to open, were found to be significantly lower in the robotic approach as compared to both laparoscopic and conventional open techniques. Despite the overall increased rate of complications combined from all the studies, the rate of significant complications such as anastomotic leaks requiring readmission and return to theater was also found to be substantially less. This study concludes that although robotic approach is in its initial stages for pelvic surgeries, it can be safely employed due to improved dexterity and visibility.

Microbes vs. Nematodes: Insights into Biocontrol through Antagonistic Organisms to Control Root-Knot Nematodes.

Root-knot nematodes (Meloidogyne spp.) are sedentary endoparasites that cause severe economic losses to agricultural crops globally. Due to the regulations of the European Union on the application of nematicides, it is crucial now to discover eco-friendly control strategies for nematode management. Biocontrol is one such safe and reliable method for managing these polyphagous nematodes. Biocontrol agents not only control these parasitic nematodes but also improve plant growth and induce systemic resistance in plants against a variety of biotic stresses. A wide range of organisms such as bacteria, fungi, viruses, and protozoans live in their natural mode as nematode antagonists. Various review articles have discussed the role of biocontrol in nematode management in general, but a specific review on biocontrol of root-knot nematodes is not available in detail. This review, therefore, focuses on the biocontrol of root-knot nematodes by discussing their important known antagonists, modes of action, and interactions.

Traditional uses, phytochemistry, pharmacology, and toxicology of the genus Artemisia L. (Asteraceae): A high-value medicinal plant.

Biologically active secondary metabolites, essential oils, and volatile compounds derived from medicinal and aromatic plants play a crucial role in promoting human health. Within the large family Asteraceae, the genus Artemisia consists of approximately 500 species. Artemisia species have a rich history in traditional medicine worldwide, offering remedies for a wide range of ailments, such as malaria, jaundice, toothache, gastrointestinal problems, wounds, inflammatory diseases, diarrhoea, menstrual pains, skin disorders, headache, and intestinal parasites. The therapeutic potential of Artemisia species is derived from a multitude of phytoconstituents, including terpenoids, phenols, flavonoids, coumarins, sesquiterpene lactones, lignans, and alkaloids that serve as active pharmaceutical ingredients (API). The remarkable antimalarial, antimicrobial, anthelmintic, antidiabetic, anti-inflammatory, anticancer, antispasmodic, antioxidative and insecticidal properties possessed by the species are attributed to these APIs. Interestingly, several commercially utilized pharmaceutical drugs, including arglabin, artemisinin, artemether, artesunate, santonin, and tarralin have also been derived from different Artemisia species. However, despite the vast medicinal potential, only a limited number of Artemisia species have been exploited commercially. Further, the available literature on traditional and pharmacological uses of Artemisia lacks comprehensive reviews. Therefore, there is an urgent need to bridge the existing knowledge gaps and provide a scientific foundation for future Artemisia research endeavours. It is in this context, the present review aims to provide a comprehensive account of the traditional uses, phytochemistry, documented biological properties and toxicity of all the species of Artemisia and offers useful insights for practitioners and researchers into underutilized species and their potential applications. This review aims to stimulate further exploration, experimentation and collaboration to fully realize the therapeutic potential of Artemisia in augmenting human health and well-being.

Molecular basis of DFNB73: mutations of BSND can cause nonsyndromic deafness or Bartter syndrome.

BSND encodes barttin, an accessory subunit of renal and inner ear chloride channels. To date, all mutations of BSND have been shown to cause Bartter syndrome type IV, characterized by significant renal abnormalities and deafness. We identified a BSND mutation (p.I12T) in four kindreds segregating nonsyndromic deafness linked to a 4.04-cM interval on chromosome 1p32.3. The functional consequences of p.I12T differ from BSND mutations that cause renal failure and deafness in Bartter syndrome type IV. p.I12T leaves chloride channel function unaffected and only interferes with chaperone function of barttin in intracellular trafficking. This study provides functional data implicating a hypomorphic allele of BSND as a cause of apparent nonsyndromic deafness. We demonstrate that BSND mutations with different functional consequences are the basis for either syndromic or nonsyndromic deafness.

Treatments for anxiety and depression in patients with chronic obstructive pulmonary disease: a literature review.

Chronic obstructive pulmonary disease (COPD) is a serious contemporary health issue. Psychological co-morbidities such as anxiety and depression are common in COPD. Current evidence for treatment options to reduce anxiety and depression in patients with COPD was examined. There is evidence available for the efficacy of pharmacological treatments, cognitive behavioural therapy, pulmonary rehabilitation, relaxation therapy and palliative care in COPD. Therapeutic modalities that have not been proven effective in decreasing anxiety and depression in COPD, but which have theoretical potential among patients, include interpersonal psychotherapy, self-management programmes, more extensive disease management programmes, supportive therapy and self-help groups. Besides pulmonary rehabilitation that is only available for a small percentage of patients, management guidelines make scant reference to other options for the treatment of mental health problems. The quantity and quality of research on mental health treatments in COPD have historically been insufficient to support their inclusion in COPD treatment guidelines. In this review, recommendations regarding assessment, treatment and future research in this important field were made.