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Human epidermal growth factor (hEGF) holds significant importance in the fields of medicine and cosmetics. Therefore, it becomes imperative to develop a highly efficient fermentation system for hEGF production. In this study, a stable hEGF-secreting expression strain was created by integrating the hEGF gene into the genome of Escherichia coli (E. coli) BL21, and an immobilized fermentation system was developed based on biofilm to facilitate continuous hEGF production. After optimization of fermentation conditions and gene dosage, the production of hEGF was increased from 13.9 mg/L to 52.4 mg/L in free-cell fermentation. Moreover, genetic modifications targeting dgcC, csgD, bcsA, and bcsB proved to enhance biofilm formation. When the bcsB was overexpressed in BL21-hEGF-C5, the biofilm-forming ability was enhanced by 91.1% and the production of hEGF was increased by 28% in biofilm-immobilized continuous fermentation. In conclusion, this study successfully confirms the feasibility of continuous hEGF production through the biofilm system of E. coli, providing valuable insights for the development of other proteins in the field of continuous biomanufacturing. [ABSTRACT FROM AUTHOR]

Abstract Background Pichia pastoris is a widely utilized host for heterologous protein expression and biotransformation. Despite the numerous strategies developed to optimize the chassis host GS115, the potential impact of changes in cell wall polysaccharides on the fitness and performance of P. pastoris remains largely unexplored. This study aims to investigate how alterations in cell wall polysaccharides affect the fitness and function of P. pastoris, contributing to a better understanding of its overall capabilities. Results Two novel mutants of GS115 chassis, H001 and H002, were established by inactivating the PAS_chr1-3_0225 and PAS_chr1-3_0661 genes involved in β-glucan biosynthesis. In comparison to GS115, both modified hosts exhibited a looser cell surface and larger cell size, accompanied by faster growth rates and higher carbon-to-biomass conversion ratios. When utilizing glucose, glycerol, and methanol as exclusive carbon sources, the carbon-to-biomass conversion rates of H001 surpassed GS115 by 10.00%, 9.23%, and 33.33%, respectively. Similarly, H002 exhibited even higher increases of 32.50%, 12.31%, and 53.33% in carbon-to-biomass conversion compared to GS115 under the same carbon sources. Both chassis displayed elevated expression levels of green fluorescent protein (GFP) and human epidermal growth factor (hegf). Compared to GS115/pGAPZ A-gfp, H002/pGAPZ A-gfp showed a 57.64% higher GFP expression, while H002/pPICZα A-hegf produced 66.76% more hegf. Additionally, both mutant hosts exhibited enhanced biosynthesis efficiencies of S-adenosyl-L-methionine and ergothioneine. H001/pGAPZ A-sam2 synthesized 21.28% more SAM at 1.14 g/L compared to GS115/pGAPZ A-sam2, and H001/pGAPZ A-egt1E obtained 45.41% more ERG at 75.85 mg/L. The improved performance of H001 and H002 was likely attributed to increased supplies of NADPH and ATP. Specifically, H001 and H002 exhibited 5.00-fold and 1.55-fold higher ATP levels under glycerol, and 6.64- and 1.47-times higher ATP levels under methanol, respectively, compared to GS115. Comparative lipidomic analysis also indicated that the mutations generated richer unsaturated lipids on cell wall, leading to resilience to oxidative damage. Conclusions Two novel P. pastoris chassis hosts with impaired β-1,3-D-glucan biosynthesis were developed, showcasing enhanced performances in terms of growth rate, protein expression, and catalytic capabilities. These hosts exhibit the potential to serve as attractive alternatives to P. pastoris GS115 for various bioproduction applications.

Background: Pichia pastoris is a widely utilized host for heterologous protein expression and biotransformation. Despite the numerous strategies developed to optimize the chassis host GS115, the potential impact of changes in cell wall polysaccharides on the fitness and performance of P. pastoris remains largely unexplored. This study aims to investigate how alterations in cell wall polysaccharides affect the fitness and function of P. pastoris, contributing to a better understanding of its overall capabilities. Results: Two novel mutants of GS115 chassis, H001 and H002, were established by inactivating the PAS_chr1-3_0225 and PAS_chr1-3_0661 genes involved in β-glucan biosynthesis. In comparison to GS115, both modified hosts exhibited a looser cell surface and larger cell size, accompanied by faster growth rates and higher carbon-to-biomass conversion ratios. When utilizing glucose, glycerol, and methanol as exclusive carbon sources, the carbon-to-biomass conversion rates of H001 surpassed GS115 by 10.00%, 9.23%, and 33.33%, respectively. Similarly, H002 exhibited even higher increases of 32.50%, 12.31%, and 53.33% in carbon-to-biomass conversion compared to GS115 under the same carbon sources. Both chassis displayed elevated expression levels of green fluorescent protein (GFP) and human epidermal growth factor (hegf). Compared to GS115/pGAPZ A-gfp, H002/pGAPZ A-gfp showed a 57.64% higher GFP expression, while H002/pPICZα A-hegf produced 66.76% more hegf. Additionally, both mutant hosts exhibited enhanced biosynthesis efficiencies of S-adenosyl-L-methionine and ergothioneine. H001/pGAPZ A-sam2 synthesized 21.28% more SAM at 1.14 g/L compared to GS115/pGAPZ A-sam2, and H001/pGAPZ A-egt1E obtained 45.41% more ERG at 75.85 mg/L. The improved performance of H001 and H002 was likely attributed to increased supplies of NADPH and ATP. Specifically, H001 and H002 exhibited 5.00-fold and 1.55-fold higher ATP levels under glycerol, and 6.64- and 1.47-times higher ATP levels under methanol, respectively, compared to GS115. Comparative lipidomic analysis also indicated that the mutations generated richer unsaturated lipids on cell wall, leading to resilience to oxidative damage. Conclusions: Two novel P. pastoris chassis hosts with impaired β-1,3-D-glucan biosynthesis were developed, showcasing enhanced performances in terms of growth rate, protein expression, and catalytic capabilities. These hosts exhibit the potential to serve as attractive alternatives to P. pastoris GS115 for various bioproduction applications. [ABSTRACT FROM AUTHOR]

Bacillus subtilis is widely employed for recombinant protein expression. B. subtilis DB104 offers a distinct advantage as a protein expression host because it is an extracellular protease-deficient derivative of B. subtilis 168. We have conducted a time-course transcriptome analysis of B. subtilis DB104 in a prior study. In the present study, we identified 10 genes that exhibited strong expression at each time point or all, based on transcriptome data. Subsequently, we assessed the strength of 12 promoters that transcribe these genes using enhanced green fluorescent protein (eGFP) as a reporter. Among these promoters, Psdp and PskfA had the highest expression levels. At 24 h, these two promoters exhibited 34.5- and 38.8-fold higher strength, respectively, than the strength of P43, the control promoter. Consequently, these two promoters were selected for further development. We enhanced these promoters by optimizing spacer length, promoter sequence, Shine–Dalgarno sequence, regulator binding sites, and terminator sequences. As a result, we successfully engineered the most potent protein expression cassette, Psdp-4, which exhibited a 3.84-fold increase in strength compared to the original Psdp promoter. Furthermore, we constructed an expression cassette for a human epidermal growth factor (hEGF) using Psdp-4 to evaluate its general application. The expression level of His tagged hEGF, quantified using ImageJ analysis and applied to SDS-PAGE, reached the highest yield of 103.9 μg/mL under the control of Psdp-4 at 24 h. The expressed hEGF protein was purified, and its bioactivity was confirmed through a cell proliferation assay using HT-29 cells. Our work demonstrates the construction of a highly efficient expression system for B. subtilis DB104 based on transcriptome data and promoter engineering. This system enables rapid, inducer-free protein expression within 24 h. It can be used as a valuable tool for various industrial applications. [ABSTRACT FROM AUTHOR]

Human epidermal growth factor (hEGF) holds significant importance in the fields of medicine and cosmetics. Therefore, it becomes imperative to develop a highly efficient fermentation system for hEGF production. In this study, a stable hEGF-secreting expression strain was created by integrating the hEGF gene into the genome of Escherichia coli (E. coli) BL21, and an immobilized fermentation system was developed based on biofilm to facilitate continuous hEGF production. After optimization of fermentation conditions and gene dosage, the production of hEGF was increased from 13.9 mg/L to 52.4 mg/L in free-cell fermentation. Moreover, genetic modifications targeting dgcC, csgD, bcsA, and bcsB proved to enhance biofilm formation. When the bcsB was overexpressed in BL21-hEGF-C5, the biofilm-forming ability was enhanced by 91.1% and the production of hEGF was increased by 28% in biofilm-immobilized continuous fermentation. In conclusion, this study successfully confirms the feasibility of continuous hEGF production through the biofilm system of E. coli, providing valuable insights for the development of other proteins in the field of continuous biomanufacturing.

Bacillus subtilis is widely employed for recombinant protein expression. B. subtilis DB104 offers a distinct advantage as a protein expression host because it is an extracellular protease-deficient derivative of B. subtilis 168. We have conducted a time-course transcriptome analysis of B. subtilis DB104 in a prior study. In the present study, we identified 10 genes that exhibited strong expression at each time point or all, based on transcriptome data. Subsequently, we assessed the strength of 12 promoters that transcribe these genes using enhanced green fluorescent protein (eGFP) as a reporter. Among these promoters, Psdp and PskfA had the highest expression levels. At 24 h, these two promoters exhibited 34.5- and 38.8-fold higher strength, respectively, than the strength of P43, the control promoter. Consequently, these two promoters were selected for further development. We enhanced these promoters by optimizing spacer length, promoter sequence, Shine–Dalgarno sequence, regulator binding sites, and terminator sequences. As a result, we successfully engineered the most potent protein expression cassette, Psdp-4, which exhibited a 3.84-fold increase in strength compared to the original Psdp promoter. Furthermore, we constructed an expression cassette for a human epidermal growth factor (hEGF) using Psdp-4 to evaluate its general application. The expression level of His tagged hEGF, quantified using ImageJ analysis and applied to SDS-PAGE, reached the highest yield of 103.9 μg/mL under the control of Psdp-4 at 24 h. The expressed hEGF protein was purified, and its bioactivity was confirmed through a cell proliferation assay using HT-29 cells. Our work demonstrates the construction of a highly efficient expression system for B. subtilis DB104 based on transcriptome data and promoter engineering. This system enables rapid, inducer-free protein expression within 24 h. It can be used as a valuable tool for various industrial applications.

Objective The aim of the study is to see the prevalence of different molecular subtypes in breast cancer patients among two different age groups: ≤40 years and >40 years. Materials and Methods Retrospective study was conducted from January 2019 to December 2019. We studied 568 cases of breast carcinoma and classified them into four molecular subtypes—luminal A, luminal B, human epidermal growth factor-2 (HER 2), and triple negative. Cases were divided into two different groups: (1) ≤40 years and (2) >40 years. Statistical Analysis was done by using SPSS software version 20.0. Results Out of 568 cases, 151 (26.6%) were ≤40 years of age and 417 (73.4%) were >40 years of age. The most common histological subtype of breast cancer was ductal carcinoma in 548 cases and the most common grade was grade III. Immunohistochemistry was done in 432 patients. In younger age group, the most common molecular subtype was luminal B (31%) followed by triple negative (20%), luminal A (14%), and then HER 2 (5.3%), while in the older age group most common molecular subtype was luminal B (27.8%) followed by triple negative (14%), HER 2 (12.2%), and then luminal A (12%). Conclusion Luminal B is found to be the most common subtype in Northeast Indian women with breast cancer, as compared with other studies in which luminal A was the most common subtype. This could be due to the reason that Ki-67 was not done in most of the other studies.

Breast cancer is a heterogeneous disease with complex molecular pathogenesis. Overexpression of several tyrosine kinase receptors is associated with poor prognosis, therefore, they can be key targets in breast cancer therapy. Tyrosine kinase inhibitors (TKIs) have emerged as leading agents in targeted cancer therapy due to their effectiveness in disrupting key molecular pathways involved in tumor growth. TKIs target various tyrosine kinases, including the human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), Vascular endothelial growth factor receptor (VEGFR), anaplastic lymphoma kinase (ALK), vascular endothelial growth factor receptor (VEGFR)-associated multi-targets, rearranged during transfection (RET), fibroblast growth factor receptor (FGFR), receptor tyrosine kinase-like orphan signal 1 (ROS1), Mitogen-activated protein kinase (MAPK), and tropomyosin receptor kinase (TRK). These drugs target the tyrosine kinase domain of receptor tyrosine kinases and play a vital role in proliferation and migration of breast cancer cells. Several TKIs, including lapatinib, neratinib, and tucatinib, have been developed and are currently used in clinical settings, often in combination with chemotherapy, endocrine therapy, or other targeted agents. TKIs have demonstrated remarkable benefits in enhancing progression-free and overall survival in patients with breast cancer and have become a standard of care for this population. This review provides an overview of TKIs currently being examined in preclinical studies and clinical trials, especially in combination with drugs approved for breast cancer treatment. TKIs have emerged as a promising therapeutic option for patients with breast cancer and hold potential for treating other breast cancer subtypes. The development of new TKIs and their integration into personalized treatment strategies will continue to shape the future of breast cancer therapy., Competing Interests: Declaration of Competing Interest The authors declared no potential conflicts of interest concerning the research, authorship, and/or publication of this article., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

ABSTRACT Objective: To compare the effect of topical epidermal growth factor (EGF) instillation versus observation alone on healing of acute tympanic membrane perforations in terms of closure and hearing test results. Methods: Design: Randomized, Open label, Clinical Trial Setting: Tertiary Government Training Hospital Participants: Seventeen (17) ENT-HNS OPD patients aged between 18 to 65 years old diagnosed with acute tympanic membrane perforation were included in the study. Group A underwent observation while group B was treated with recombinant human EGF solution. Follow- up was on a weekly basis (7th, 14th, 21st and 28th days) where video otoscopy for documentation and measurement of perforation using ImageJTM software was done. Pure tone audiometry was used to compare hearing improvement pre and post study in both observation and treatment groups. Results: At baseline, there was no significant difference in the sizes of perforations: 24.20 ± 9.95 (treatment) vs. 32.64 ± 11.62 (observation) with a p-value of .131. Following treatment, mean changes in perforation size were significantly greater in the treatment group compared to the observation group from baseline to day 7 (M = -9.08, n = 15.11 vs. M = -1.06, n = 31.58); p = .009; day 7 to 14 (M = -6.37, n = 13.78 vs. M = -0.79, n = 30.79); p = .003; and from day 14 to 21 (M = -5.65, n = 10.89 vs. M = -0.72, n = 30.07); p = .004 but not from day 21 to 28 (M = -4.16, n = 13.99 vs. M = -0.36, n = 29.71; p = .021. From baseline pure tone averages, four participants with mild hearing loss and two with moderate hearing loss achieved normal hearing in the treatment group (while one each with moderate and severe hearing loss did not improve). None of the observation group participants had improved hearing. Conclusion: Based on our limited experience, topical EGF can be used for traumatic tympanic membrane perforation and otitis media with dry ear perforation during the acute phase or within 3 months of perforation.

Human epidermal growth factor (hEGF) is a key factor involved in wound healing owing to its powerful ability to stimulate cell proliferation. In this study, we used piggyBac transposon technology to produce transgenic silkworms expressing the hEGF protein fused to truncated heavy chain (FibH-hEGF). The FibH–hEGF fusion protein was successfully expressed and secreted into silkworm cocoons. Compared to wild-type silk, the transgenic silkworm silk had the similar morphology about silks fiber surface and cocoon nets, while the secondary structure between the transgenic silk and wild-type silk was different. Most importantly, transgenic silkworm cocoon silk powder extract significantly increased human fibroblast FIB cell proliferation for a long duration with no apparent cytotoxicity. Our study provides a promising method for obtaining cost-effective and functional biomaterials for the fabrication of wound dressings. [ABSTRACT FROM AUTHOR]

PurposeTo compare the efficacy and safety between pyrotinib (Pyr) and trastuzumab emtansine (T-DM1) in pre-treated human epidermal growth factor receptor 2-positive (HER2+) metastatic breast cancer (MBC) patients.MethodsA comprehensive literature search of the PubMed, EMBASE, and Web of Science was performed in August 2020. Randomized clinical trials comparing the efficacy and safety between different anti-HER2 regimens in patients pre-treated with trastuzumab (Tra) and a taxane in metastatic settings (≤second-line treatment) were included. A fixed effects network meta-analysis based on the Bayesian inferential framework was conducted for progression-free survival (PFS), overall survival (OS), overall response rate (ORR), and grade ≥3 adverse events (AEs). Values of surface under cumulative ranking probability curve (SUCRA) were calculated to offer a ranking of all regimens.ResultsTwelve studies with 4,353 subjects were identified. Nine regimens were included into the network: T-DM1, lapatinib-capecitabine (Lap-Cap), Tra-Cap, Cap, neratinib (Ner), pertuzumab (Per)-Tra-Cap, Pyr-Cap, atezolizumab (Ate)-T-DM1, and Ner-Cap. For PFS, Pyr-Cap was more favorable than T-DM1 (hazard ratio, 95% confidence interval: 0.77, 0.70–0.86), Lap-Cap (0.64, 0.59–0.69), Tra-Cap (0.63, 0.56–0.70), Cap (0.50, 0.45–0.56), Ner (0.59, 0.51–0.69), Per-Tra-Cap (0.68, 0.59–0.79), and Ner-Cap (0.72, 0.64–0.81). For OS, Pyr-Cap showed further improvement than Lap-Cap (hazard ratio, 95% confidence interval: 0.71, 0.52–0.99), Cap (0.68, 0.49–0.96), and Ner (0.65, 0.45–0.94). For ORR, Pyr-Cap was significantly superior than Cap (odds ratio, 95% confidence interval: 7.87, 1.22–56.51). No significant difference was observed in grade ≥3 AEs among all the regimens. Pyr-Cap ranked in the highest in PFS, OS, ORR, and grade ≥3 AEs (SUCRA = 99.4, 89.7, 86.4, and 89.3%).ConclusionsThese results indicate that Pyr may be more effective than T-DM1 in HER2+ MBC patients pre-treated with Tra and a taxane. However, it may be associated with more grade ≥3 AEs.

Purpose: To compare the efficacy and safety between pyrotinib (Pyr) and trastuzumab emtansine (T-DM1) in pre-treated human epidermal growth factor receptor 2-positive (HER2+) metastatic breast cancer (MBC) patients. Methods: A comprehensive literature search of the PubMed, EMBASE, and Web of Science was performed in August 2020. Randomized clinical trials comparing the efficacy and safety between different anti-HER2 regimens in patients pre-treated with trastuzumab (Tra) and a taxane in metastatic settings (≤second-line treatment) were included. A fixed effects network meta-analysis based on the Bayesian inferential framework was conducted for progression-free survival (PFS), overall survival (OS), overall response rate (ORR), and grade ≥3 adverse events (AEs). Values of surface under cumulative ranking probability curve (SUCRA) were calculated to offer a ranking of all regimens. Results: Twelve studies with 4,353 subjects were identified. Nine regimens were included into the network: T-DM1, lapatinib-capecitabine (Lap-Cap), Tra-Cap, Cap, neratinib (Ner), pertuzumab (Per)-Tra-Cap, Pyr-Cap, atezolizumab (Ate)-T-DM1, and Ner-Cap. For PFS, Pyr-Cap was more favorable than T-DM1 (hazard ratio, 95% confidence interval: 0.77, 0.70–0.86), Lap-Cap (0.64, 0.59–0.69), Tra-Cap (0.63, 0.56–0.70), Cap (0.50, 0.45–0.56), Ner (0.59, 0.51–0.69), Per-Tra-Cap (0.68, 0.59–0.79), and Ner-Cap (0.72, 0.64–0.81). For OS, Pyr-Cap showed further improvement than Lap-Cap (hazard ratio, 95% confidence interval: 0.71, 0.52–0.99), Cap (0.68, 0.49–0.96), and Ner (0.65, 0.45–0.94). For ORR, Pyr-Cap was significantly superior than Cap (odds ratio, 95% confidence interval: 7.87, 1.22–56.51). No significant difference was observed in grade ≥3 AEs among all the regimens. Pyr-Cap ranked in the highest in PFS, OS, ORR, and grade ≥3 AEs (SUCRA = 99.4, 89.7, 86.4, and 89.3%). Conclusions: These results indicate that Pyr may be more effective than T-DM1 in HER2+ MBC patients pre-treated with Tra and a taxane. However, it may be associated with more grade ≥3 AEs. [ABSTRACT FROM AUTHOR]

Human epidermal growth factor (hEGF) has gained clinical importance due to its ability to promote wound healing. Due to its commercial applications and high market demand, recombinant EGF has been produced in several forms. Currently, plant expression system is considered as potential alternative for low-cost recombinant protein production. Hence, this study focused on improving the production of hEGF in plants by effective gene construct design and optimizing the Agrobacterium culture conditions for high protein production. In this context, hEGF gene was cloned into plant geminiviral expression vector pBYR2e and transformed in to N. benthamiana leaves via., agroinfiltration. The recombinant hEGF was purified from the plant crude extracts by single-step affinity chromatography. Furthermore, the plant-produced hEGF has shown to promote cell migration comparable to commercial hEGF in HaCaT cells in vitro. These results indicated the potential of plant expression system for the production of recombinant hEGF for tissue engineering applications.

Introduction: Breast cancer is leading cancer in women, and the incidence of breast cancer in India is on the rise. The most common histologic type of breast cancer is infiltrating ductal carcinoma. Prognostic and predictive factors are used in the management of breast cancer. Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2/neu) are immunohistochemical markers of prognosis as well as predictors of response to therapy. Aims and Objectives: The study was conducted to evaluate ER, PR, and HER2/neu expressions in invasive ductal carcinomas of the breast by immunohistochemistry, to explore the correlation of these markers to each other and to various clinicopathological parameters: age of the patient, histological grade, tumor size, and lymph node metastasis. Materials and Methods: This prospective study was conducted on 100 cases of infiltrating ductal carcinoma. Slides were prepared from blocks containing cancer tissue, and immunohistochemical staining was done for ER, PR, and HER2/neu expressions. Interpretation of expressions was done using Allred scoring system for ER/PR and the American Society of Clinical Oncology/College of American Pathologists guidelines for HER2/neu. Statistical analysis was performed to determine the statistical significance by applying Chi-square test. Results: Majority of tumors were ER and PR positive and HER2/neu negative. ER and PR correlated significantly with age, tumor size, and tumor grade; whereas, HER2/neu correlated significantly with tumor size only. No association was seen with axillary lymph node metastasis. ER and PR expression correlated with each other, but none correlated with HER2/neu. Conclusions: As the majority of the tumors are ER, PR positive and since ER and PR correlate with each other as well as with age, tumor size, and grade. Therefore, routine assessment of hormone receptors is recommended for prognostic and therapeutic information in breast cancer cases.

To validate the efficacy of recombinant human epidermal growth factor (hEGH) in healing diabetic foot ulcers (DFUs) at biochemical and molecular levels. A total of 50 noninfected DFU subjects were recruited for the study and divided into 2 groups based on the treatment application on the subjects. Group 1: DFU subjects treated with hEGH gel-based product called Regen-D 150 (n = 27) and group 2: DFU subjects treated with alternative placebo as the control group (n = 23). Patients were observed for 30 days and punch biopsy was taken at days 0 and 14. Histologic analysis was done to study the matrix alignment, cellular infiltration, and differentiation of epithelial layers. Biochemical analysis was done to quantitatively estimate the amount of collagen and proteoglycans regenerated in the wound area. Complete healing of ulcers was observed in 21 (78%) subjects in group 1, whereas only 12 (52%) subjects among group 2 reported of complete healing of ulcer after completion of the study period of 30 days. Collagen and fibroblasts were significantly developed in group 1 when observed in the follow-up samples. Healing time of the wound among the group 1 subjects was significantly less than the group 2 subjects (45 ± 12 vs 72 ± 18 days, P <.0001) and even showed a better blood glucose level. Early and regular application of the hEGH on DFUs will lead to prevention of leg amputations and would serve to act as a major treatment therapy for healing of chronic wounds. [ABSTRACT FROM AUTHOR]

• High yield of hEGF can be achieved by the guidance of xynD SP in B. subtilis. • The hEGF production of B. subtilis PT5 bearing two T7 expression cassette could be a suitable replacement for IPTG induction. • hEGF can be harvested with high purity by the His-tagged affinity purification. Human epidermal growth factor (hEGF) is a crucial factor in wound healing, and therefore has wide applications in the pharmaceutical and cosmetics industries. To improve the production of hEGF protein for the industrial scale, we established an hEGF gene construct for recombinant secretion by Bacillus subtilis. Examination of the secretion abilities of various signal peptides (SPs) revealed six SPs that could effectively guide hEGF secretion from B. subtilis into the culture medium. The highest production level was obtained when the hEGF gene was fused with the xynD SP (lipo type). Two copies of the hEGF expression cassette had the same effect on improving production yield as isopropyl-β- d -1-thiogalactopyranoside (IPTG) induction, and the highest yield of hEGF can reach 360 ± 9.41 mg/L. Moreover, high-purity hEGF was obtained using a His-tagged purification system. Overall, these results demonstrate that B. subtilis is a suitable cell factory for hEGF production with improved yield compared to previous systems. [ABSTRACT FROM AUTHOR]

Human epidermal growth factor (hEGF) has been known to have excellent wound-healing activity. However, direct application to the wound area can lead to low hEGF bioavailability due to protease enzymes or endocytosis. The use of liposomes as coatings and carriers can protect hEGF from degradation by enzymes, chemical reactions, and immune reactions. Sustained release using a matrix polymer can also keep the levels of hEGF in line with the treatment. Therefore, this study aimed to develop a film-forming spray of water-soluble chitosan (FFSWSC) containing hEGF-liposomes as a potential wound dressing. The hEGF-liposomes were prepared using the hydration film method, and the preparation of the FFSWSC was achieved by the ionic gelation method. The hydration film method produced hEGF-liposomes that were round and spread with a Z-average of 219.3 nm and encapsulation efficiency of 99.87%, whereas the film-forming solution, which provided good sprayability, had a formula containing 2% WSC and 3% propylene glycol with a viscosity, spray angle, droplet size, spray weight, and occlusion factor of 21.94 ± 0.05 mPa.s, 73.03 ± 1.28°, 54.25 ± 13.33 µm, 0.14 ± 0.00 g, and 14.57 ± 3.41%, respectively. The pH, viscosity, and particle size of the FFSWSC containing hEGF-liposomes were stable during storage for a month in a climatic chamber (40 ± 2 °C, RH 75 ± 5%). A wound healing activity test on mice revealed that hEGF-liposomes in FFSWSC accelerated wound closure significantly, with a complete wound closure on day 6. Based on the findings, we concluded that FFSWSC containing hEGF-liposomes has the potential to be used as a wound dressing.

Objective: Aim: To explore efficacy and safety of combined anti-tumor treatments against breast cancer to help health professionals and decision-makers take strategies to slow the spread of breast cancer and improve women's health., Patients and Methods: Material and Methods: A cross-sectional study was used by various Iraqi governments. The survey was conducted between July 1, 2022 and April 30, 2023. The study depended on describing studying of 100 patients in detail and with long-term follow-up who go to hospitals and take anti-cancer medications from different provinces of central and south Iraq. The questionnaire form contains 17 fields divided into three sections. The diagnosis tumor before and after treatment parameters: histopathology, CT-scan, tumor marker (Nuclear protein Ki67, Cancer antigen 153 (CA 153), human epidermal growth factor receptor 2, and carcinoembryonic antigen), renal function tests, liver function tests, and Complete Blood Count., Results: Results: The use of anti-tumor medications was coupled with markedly decreased tumor cell proliferation via reduced biomarker levels of cancer (CA-15-3), HER-2, and Ki-67 levels, and decreased the mammary ductal epithelium's thickness., Conclusion: Conclusions: The study showed efficiency of combination medications to prevent breast cancer cell development by preventing cell growth.

Transgenic silkworm expression systems have been applied for producing various recombinant proteins. Knocking out or downregulating an endogenous silk protein is considered a viable strategy for improving the ability of transgenic expression systems to produce exogenous proteins. Here, we report the expression of human epidermal growth factor (hEGF) in a P25 gene knockout silkworm. The hEGF gene regulated by the P25 gene promoter was integrated into a silkworm’s genome. Five transgenic positive silkworm lineages were generated with different insertion sites on silkworm chromosomes and the ability to synthesize and secrete proteins into cocoons. Then, a cross-strategy was used to produce transgenic silkworms with a P25 gene knockout background. The results of the protein analysis showed that the loss of an endogenous P25 protein can increase the hEGF production to about 2.2-fold more than normal silkworms. Compared to those of transgenic silkworms with wild type (non-knockout) background, the morphology and secondary structure of cocoon silks were barely changed in transgenic silkworms with a P25 gene knockout background, indicating their similar physical properties of cocoon silks. In conclusion, P25 gene knockout silkworms may become an efficient bioreactor for the production of exogenous proteins and a promising tool for producing various protein-containing silk biomaterials.

Mesenchymal stem cells (MSCs) repair tissue injury by upregulating the paracrine secretion of cytokines and growth factors. Human MSC has been recognized as a promising therapeutic material for treatment of various human diseases. Even though the effect of epidermal growth factor (EGF) has been well investigated, the synergetic effect of EGF and MSC has not been studied. Therefore, we expect our basic study to contribute to developing new therapeutic reagents for skin diseases or innovative cosmetics. In this study, we examined the effect of human epidermal growth factor-transfected MSCs (hEGF MSCs) on human keratinocyte HaCaT cell proliferation and the mechanisms that regulate matrix metalloproteinase (MMP)-1 expression in HaCaT cells. To identify the hEGF plasmid and its transfection into MSCs, we performed gel electrophoresis and quantitative PCR. Proliferation and migration of HaCaT cells were examined using water Soluble Tetrazolium (WST-1) and wound-healing assays, respectively. Zymography was performed to investigate the correlation between hEGF MSC-conditioned medium (CM)-treated HaCaT cells and MMP-1 expression. We found that cell proliferation and wound-healing rates were increased in hEGF MSC-CM-treated HaCaT cells compared to those in MSC-CM-treated cells, and conversely collagenase activity was decreased. The mRNA and protein levels of MMP-1 were also decreased in hEGF MSC-CM-treated HaCaT cells. 2-DE analysis showed that the expression of carboxypeptidase, which promotes growth factors and wound healing, was increased in hEGF MSC-CM-treated HaCaT cells. Finally, western blot was used to determine whether MMP-1 expression was reduced via the mitogen-activated protein kinase (MAPK) pathway; the results showed that the levels of MAPK pathway-related proteins (pErk, pJNK, and p-p38) and the levels of transcription factors (pCREB, NFκB, and p-c-Fos) were decreased. In addition, pAkt expression was found to be elevated. The results of our study suggest that hEGF MSCs promote cell proliferation and reduce MMP-1 expression via the MAPK pathway in human keratinocyte HaCaT cells. • Mesenchymal stem cells (MSCs) are multipotent progenitor cells that can be differentiated. • Examined the effect of human epidermal growth factor-transfected MSCs (hEGF MSCs) on human keratinocyte. • MSC-conditioned medium (MSC-CM), which contains various cytokines and growth factors, are safer than MSCs. • Investigated therapeutic effect of hMSCs overexpressing hEGF on human keratinocytes HaCaT cells. • Upregulation of carboxypeptidase affects HaCaT cell proliferation induced by hEGF MSCs. [ABSTRACT FROM AUTHOR]

Background: Human epidermal growth factor (hEGF) has drawn intense research attention due to its potential ability to promote healing of serious injuries, such as cuts, burns, and diabetic ulcers. Although hEGF displays prospective clinical value, the growth factor is restricted to the treatment of chronic diabetic ulcers because of its high production cost. Methods: Leguminous plant peanut (Arachis hypogaea L.) hairy roots contain relatively few toxic and harmful substances, and tested as an excellent production system for hEGF in our study. To explore the possibility of hEGF expression in peanut, hEGF overexpression hairy roots were obtained by infecting leaves with Agrobacterium rhizogenes R1601. Results: The maximum transgenic hairy roots inducing rate was 82%. Protein purification and mass spectrometry assays showed that the protein expressed in peanut hairy roots was identified as hEGF. Furthermore, Methylthiazolyldiphenyl-tetrazolium bromide assay showed that hEGF promoted HL-7702 liver cells proliferation, which indicate that hEGF has biological activity and non-toxic on human cells. Conclusion: Our results demonstrate the capacity of peanut hairy root cultures as a controlled, sustainable, and scalable production system that can be induced to produce valued human proteins, such as hEGF.

Trastuzumab has been widely applied as a treatment for human epidermal growth factor 2 (HER2)- overexpressing breast cancer. However, the therapeutic efficacy of trastuzumab is limited. Flap endonuclease 1 (FEN1) is a multifunctional endonuclease that has a crucial role in DNA recombination and repair. Inhibition of FEN1 is associated with the reversal of anticancer drug resistance. However, it is unclear whether FEN1 is involved in trastuzumab resistance. In the present study, it was demonstrated that trastuzumab increases the expression of FEN1, and FEN1 knockdown significantly enhanced the sensitivity of BT474 cells to trastuzumab (P<0.05). It was also revealed that trastuzumab induced HER receptor activation, increased binding with FEN1 and estrogen receptor a (ERα), and upregulated ERα-target gene transcription (P<0.05). Upon silencing of FEN1 expression with siRNA, activation of HER receptor and FEN1 binding to ERα were decreased, and trastuzumab-induced ERα target gene upregulation was partially ameliorated (P<0.05). These results suggest that FEN1 may mediate trastuzumab resistance via inducing HER receptor activation and enhancing ERα-target gene transcription. The findings of the present study indicate a novel role of FEN1 in trastuzumab resistance, suggesting that targeting FEN1 may enhance the efficiency of trastuzumab as a treatment for HER2-positive breast cancer. [ABSTRACT FROM AUTHOR]

Introduction: Breast cancer (BC), a heterogeneous disease, is one of the leading causes of cancer-related deaths among women worldwide. Circulating cell-free DNA (cfDNA) levels have been persistently reported to be elevated in BC patients. In the current study, we evaluated the correlation between the cfDNA levels in patients with BC and its subtypes., Methods: We recruited newly diagnosed, histopathologically confirmed BC patients aged >18 years (N=39), who did not have any previous malignancy, from the Department of Surgical Oncology, Indira Gandhi Institute of Medical Sciences (IGIMS), Patna, Bihar, India. A total of 6 ml of venous blood was collected from each subject; of this, 1 ml was subjected to complete blood count (CBC), and 4 ml was transferred to a clot-activated collection vial for plasma separation and the cfDNA isolation thereof. In addition to the basic demographic history of each patient, the information on the cancer subtype was as also recorded from the medical records of each patient. All the data were analysed by GraphPad Prism Version 8 (Insightful Science, LLC, San Diego, California, United States). One-way ANOVA was used to test the difference between more than two groups. Pearson correlation was also estimated between cfDNA levels and various CBC indices. A two-tailed p-value<0.05 was considered statistically significant., Results: The mean age of included patients was 48.6±8.20 years. The mean levels of cfDNA were 2.81±2.39 ng/µL. The mean counts of various blood cell types and other indices of CBC were in the normal range. Compared to BC patients with estrogen receptors (ER+), the cfDNA levels were significantly higher in patients with human epidermal growth factor receptor 2 (HER2+) and triple-negative BC (TNBC) (p<0.05).  Conclusion: The elevated levels of cfDNA in patients with BC can be a prognostic marker for the disease subtype. However, more replicative studies are warranted to substantiate our findings., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, P et al.)

Se realizó un estudio descriptivo y transversal de 120 pacientes con úlceras del pie diabético, atendidos en el Centro de Diagnóstico Integral "El Bajo" perteneciente al municipio San Francisco, Maracaibo, Estado de Zulia, República Bolivariana de Venezuela, desde febrero de 2011 hasta igual mes de 2012, con vistas a evaluar la efectividad de la administración del factor de crecimiento epidérmico humano. En la casuística predominaron el grupo etario de 55-59 años y el sexo femenino. En todos los integrantes de la serie se logró la granulación y la cicatrización de las lesiones y no fue necesario efectuar la amputación. La administración intralesional del Heberprot-P® puede completar el cierre de la lesión, por lo cual es un medicamento seguro y conveniente para sanar dichas úlceras.A descriptive and cross-sectional study was conducted in 120 patients with diabetic foot ulcers treated at "El Bajo" Comprehensive Diagnosis Center belonging to San Francisco municipality, Maracaibo, State of Zulia, Bolivarian Republic of Venezuela, from February 2011 to February 2012 in order to evaluate the effectiveness of the administration of human epidermal growth factor. Age group of 55-59 years and the females prevailed in the case material. In all patients of the series granulation and healing of lesions was obtained and amputation was not necessary. Intralesional administration of Heberprot-P® can complete closure of the lesion; therefore it is a safe and advisable drug to heal these ulcers.

The authors convey their thoughts about the importance of receptor status discordance between breast cancer primary and brain metastasis. Topics mentioned include an increase in the cumulative incidence of brain metastases in breast cancer, mean age at diagnosis of primary breast cancer, and univariate analysis of the effect of patient and tumor attributes on survival after brain metastasis.