Mahmood Rasool^1*, Khalid I Alhassan², Sajjad Karim¹, Absarul Haque³, Mohammed HZ Mutwakil², Mohammed Alharthi⁴, Adeel G Chaudhary¹, Peter Natesan Pushparaj¹

¹Center of Excellence in Genomic Medicine Research, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia

²Department of Biological Science, King Abdulaziz University, Jeddah, Saudi Arabia

³King Fahd Medical Research Center, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia

⁴Faculty of Medicine, King Abdulaziz University Hospital, King Abdulaziz University, Jeddah, Saudi Arabia

Abstract

Despite considerable advancements in research, particularly in oncology, colorectal cancer (CRC) remains a formidable and deadly disease. It is crucial to delve deeper into the effects of targeted therapies, signaling pathways, and genetic regulation to ensure the effectiveness of cancer treatments. In this study, we obtained microarray data for four patient-derived CRC organoids from the Gene Expression Omnibus (GEO) database (accession number: GSE114060). We then used several next-generation knowledge discovery (NGKD) tools, such as GEO2R, Metascape, WebGestalt, and Ingenuity Pathway Analysis (IPA) software, to investigate the underlying molecular mechanisms in colorectal cancer-derived organoids treated with trametinib compared to those treated with DMSO. Our NGKD analysis revealed upregulation of SAMD, TP53, and SPTLC3 and downregulation of SPRED1, TGFBI, and DUSP6. The MAPK/ERK signaling pathway was significantly downregulated and was associated with reduced expression of CDX2 in CRC organoids treated with trametinib. We concluded that SAMD9, SPRED1, TGFBI, TP53, and DUSP6 are differentially regulated and can play a pivotal role in the downregulation of the MAPK/ERK signaling pathway to suppress CRC. The use of targeted therapies to regulate the specific gene signatures identified in the current study may be beneficial in the CRC associated tumor suppression . On the other hand, upregulation of SPTLC3 may be induced by MEK inhibitors and may cause hepatotoxicity alongside nonalcoholic steatohepatitis.

Keywords: Colorectal cancer, Organoids, Tumor suppression, MAPK/ERK signaling pathway, MEK inhibitor, Hepatotoxicity

Mahmood Rasool^1*, Khalid I. Alhassan², Sajjad Karim¹, Absarul Haque³, Mohammed H.Z. Mutwakil², Mohammed Alharthi⁴, Adeel G. Chaudhary¹, Peter Natesan Pushparaj¹

¹Center of Excellence in Genomic Medicine Research, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia

²Department of Biological Science, King Abdulaziz University, Jeddah, Saudi Arabia

³King Fahd Medical Research Center, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia

⁴Faculty of Medicine, King Abdulaziz University Hospital, King Abdulaziz University, Jeddah, Saudi Arabia

Abstract

The aim of our research was to identify molecular targets that can be targeted by drugs and patient-specific models for personalized medicine for colorectal cancer (CRC). Here, we obtained high-throughput RNA sequencing data from Gene Expression Omnibus (GEO) with accession number GSE205787 and analyzed it using next-generation knowledge discovery tools such as BioJupies and Ingenuity Pathway Analysis (IPA) software. Differentially expressed genes (DEGs) were identified by comparing the raw counts from 47 CRC patient-derived spheroids (CRC-CSCs) with those from normal spheroids from the epithelium of the colon and rectum of healthy individuals, using BioJupies tools. IPA was used to identify differentially regulated canonical pathways, upstream regulators of CRC, non-directional networks, diseases, and biofunctions, as well as to conduct subsequent perturbation analysis using the Molecular Prediction Analysis (MAP) tool. Our study demonstrates that several KEGG pathways, including the AMPK, Phospholipase D, MAPK, and PI3-AKT signaling pathways, were significantly downregulated in the CRC-CSC group. Additionally, Wnt signaling and FGFR pathways were significantly upregulated. Moreover, according to Wikipathways, the EGF/EGFR signaling pathway, MAPK signaling pathway, G-protein signaling pathway, and Focal Adhesion-PI3-AKT pathway were downregulated in the CRC-CSC group. Furthermore, based on the Reactome, the Metabolism, Vesicle-mediated transport, RAF signaling, and G-alpha (12/13) signaling pathways were also downregulated in the CRC-CSC group. Utilizing innovative drug combination approaches and innovative drug delivery techniques, CRC treatments can be enhanced by modulating the FGFR, EGFR, and AMPK signaling pathways, which may ultimately lead to improved patient outcomes.

Keywords: Colorectal cancer, Cancer stem cell spheroids, AMP‐activated protein kinase signaling, Fibroblast Growth Factor Receptor, Epidermal Growth Factor Receptor, BioJupies, Ingenuity Pathway Analysis

Narumon Tangthirasunun^1*, Darbhe Jayarama Bhat^2,3 , Supattra Poeaim¹

¹Department of Biology, School of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, 10520, Thailand

²Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh, 11451, Saudi Arabia

³Vishnugupta Vishwavidyapeetam, Ashoke, Gokarna, 581326, India

Abstract

Twenty-seven coprophilous fungi, isolated from field-fed cow dung in an organic farm in Thailand, were identified using morphology and ITS barcode. A total of five genera viz. Aspergillus, Hamigera, Paecilomyces, Penicillium, and Talaromyces were identified with varying numbers and growth rates. These fungi were evaluated for their antibacterial properties against Gram-positive (Bacillus subtilis, Kocuria rhizophila, Staphylococcus aureus and St. epidermidis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. Pe. javanicum NTD-SP2-01 and Talaromyces sp. NTD-SP5-48 exhibited activity against all bacteria when tested with agar plug diffusion method. Talaromyces sp. NTD-SP5-48 was particularly effective against Gram-negative bacteria. As. terreus NTD-NG1-05 displayed the highest activity against five bacterial strains, except Ps. aeruginosa. Notably, As. terreus NTD-NG1-05 and Talaromyces sp. NTD-SP5-48 demonstrated extended antibacterial activity in the agar disk diffusion method, with fermented broth (FB) showing superior inhibitory effects compared to mycelial extract (MY). Both isolates demonstrated significant antibacterial activity against B. Subtilis. Furthermore, all isolates exhibited significant antibacterial activity against B. subtilis, with a diffusion of 0.125 mg/disk. Only Talaromyces sp. NTD-SP5-48 (FB) displayed the highest inhibition activity against Ps. aeruginosa, with a diffusion of 1 mg/disk (100 mg/mL). In terms of enzyme activity, all isolates exhibited cellulase activity, with Talaromyces sp. showing the highest cellulase activity, followed by As. terreus. Laccase activity was only observed in the unidentified isolate NTD-SP5-34, while none of the isolates showed pectinase activity.

Keywords: Antimicrobial activity, Coprophilous fungi, Heat-resistant fungi, Lignocellulolytic enzymes

Masood Ur Rehman¹, Syed Muhammad Suhail¹, Iqbal Munir², Rajwali Khan^1*

¹Department of Livestock Management, Breeding and Genetics, The University of Agriculture, Peshawar, 25130, Khyber Pakhtunkhwa, Pakistan

²Institute of Biotechnology and Genetic Engineering, The University of Agriculture, Peshawar, 25130, Khyber Pakhtunkhwa, Pakistan

Abstract

The current study aimed to evaluate the impact of three different agroecological zones on the productive and reproductive performance of Holstein-Friesian (HF) cows, along with their comparative analysis of physiological and molecular markers under subtropical conditions of Pakistan. The productive (milk yield and composition) and reproductive (service per conception and calving interval) performance of genetically identical (n=210) HF cows placed across three agroecological zones: irrigated lowlands (Okara), wet (Abbottabad), and dry (Queta) highlands were analyzed. Additionally, heat stress markers (superoxide dismutase, glutathione peroxidase, serum cortisol, bovine heat shock protein 70 (HSP-70), and blood glucose) from five cows in each location (n=15 in total) were also investigated in early and late summers using commercial ELISA and calorimetric kits. Furthermore, the mRNA levels of heat shock protein genes HSPA8 and HSP90AB1 were also quantified in three agroecological zones through qRT-PCR.  The results revealed that cows raised in the wet highlands of Abbottabad exhibited significantly higher (P<0.05) milk production (daily and total lactational yield), and the lowest service period, calving interval and number of services per conception as compared to the cows from the other two agroecological zones. Furthermore, there was a significant effect (P<0.05) of agroecological zones on the heat stress indicators (SOD, GPX, cortisol, blood glucose, and HSP70) profiles and expression of heat shock protein genes in HF cows. The dairy cows from Abbottabad (Wet highlands) showed a significantly lower profile of heat stress indicators as compared to the cows from the other two agroecological zones. It can be inferred that HF cows function better in moderate agroecological zones of subtropical countries.

Keywords: Heat stress indicators, Subtropics, Holstein-Friesian cows, Agroecological zone

Heer Baloch¹, Irfan Ali Sabir², Saadullah Khan Leghari¹, Muhammad Sohail Saddiq^3*, Pravej Alam⁴, Shahbaz Khan^5*, Esha Mehik Fatima⁶, Mateen Sajid⁷, Muhammad Hammad Raza⁸, Muhammad Arif Hussain¹, Muhammad Ayoub¹, Rashid Iqbal⁹

¹Department of Botany, Ghazi University, Dera Ghazi Khan, Pakistan

²College of Horticulture, South China Agricultural University, Guangzhou, 510642, China

³Department of Agronomy, Ghazi University, Dera Ghazi Khan, Pakistan

⁴Department of Biology, College of Sciences and Humanities, Prince Sattam Bin Abdulaziz University, Al- Kharj 11942, Saudi Arabia

⁵Colorado Water Center, Colorado State University, Fort Collins, CO, USA

⁶Department of Entomology, Muhammad Nawaz Shareef University of Agriculture, Multan, Pakistan

⁷Department of Horticulture, Ghazi University, Dera Ghazi Khan, Pakistan

⁸Department of Agri. Extension and Education, Ghazi University, Dera Ghazi Khan, Pakistan

⁹Department of Agronomy, Faculty of Agriculture and Environment, The Islamia University of Bahawalpur, Pakistan

Abstract

Moringa leaf extract (MLE) as a biostimulant has demonstrated success in boosting the productivity of several agronomic crops, but its impact on Buckwheat crops remains unexplored. Buckwheat, recognized as an essential nutritional and functional food crop, often exhibits lower yields compared to major cereal crops grown in similar environments. Therefore, this research aimed to investigate the impact of different concentrations (1%, 2%, and 3%) of MLE on the agricultural performance of common buckwheat (CB) and tartary buckwheat (TB). A pot experiment was carried out according to completely randomized design with factorial arrangements having three replications. Results demonstrated significant improvements in growth parameters (branches, leaves, nodes, and internodes) for MLE-treated plants compared to the control group. Foliar treatment MLE 2% also increased chlorophyll content, improved membrane stability index (MSI) and relative water content (RWC), and enhanced biochemical composition (phenolic compounds, free amino acids, leaf proline, and soluble sugars) in both buckwheat genotypes followed by MLE 3% and MLE 1%. TB produced significantly higher grain yield (0.74 g) as compared to CB (0.43 g). The findings showed that a foliar treatment of MLE 2% led to increased grain yield in both TB (0.97 g) and CB (0.55 g) as compared to control group plants (0.37g TB and 0.22 g CB) respectively. This increase was associated with elevated activities of photosynthetic pigments, phenolic content, RWC, free amino acids, soluble sugars, and catalase in both buckwheat genotypes. In conclusion, MLE application at 2% significantly boosted the agriculture performance of buckwheat, and this study unlocked new insights into optimizing the productivity of the vital food crop.

Keywords: Biostimulant, Free amino acid, Phenolic, Buckwheat, Foliar application, Grain yield

Aeman Jilani¹, Mirza Imran Shahzad^1*, Muhammad Mohsin Zaman¹, Areeba Yousaf¹, Gildardo Rivera²

¹Institute of Biochemistry, Biotechnology and Bioinformatics, The Islamia University of Bahawalpur, Pakistan

2Laboratorio de Biotecnología Farmacéutica, Centro de Biotecnología Genómica, Instituto Politécnico Nacional, México

Abstract

Tuberculosis (TB), the white plague of Europe is still uncontrolled and fatal in many parts of the world including Pakistan. It is a major cause of morbidity and mortality in human and domestic animals in Pakistan. No new vaccine in the last hundred years has been developed except for a few encouraging results from recombinant and DNA vaccines in the past two decades. Five Mycobacterium specific genes (Rv0379, Rv3914, Rv3006, Rv0432+SP, and Rv0432-SP) were selected to develop DNA vaccine(s). All the constructs were tested on mice using both naked DNA and prime-boost methodologies. Forty-five BALB/c mice were divided into three main groups; DNA vaccine group, BCG Prime boost group, and Control group. Post-vaccine (PV) and post-challenge (PC) immune responses were confirmed through cytokine ELISA and qRT PCR. IFN-γ was additionally checked in plasma as well. Based on cytokine ELISA PC immune responses showed significant differences in TNF-α levels for both naked DNA vaccine groups (Rv0379, Rv3006, and Rv0432-SP) and BCG primed Rv3914 group in comparison to the BCG control group (p<0.05). Based on qRT PCR, IL-6, TNF-α, IFN-γ, and IL-1β showed no significant difference among all the vaccines and BCG control groups (CT range 25- 30). IFN-γ levels in plasma were analyzed PC; two vaccines Rv3006/LppZ and BCG primed Rv0432/SodC-SP (highest mean value 1360.35 pg/ml) have shown significant results (cutoff value 21pg/ml) at 63 days. All the vaccine construct(s) alone or in combination have significant therapeutic effects in comparison to BCG and negative control groups.

Keywords: Mycobacterium tuberculosis, DNA vaccine, Tumor necrosis factor-alpha, Interleukin-6, Interferon-gamma, Interleukin-1beta, Bacille Calmette-Guérin (BCG)

Tayyaba Asghar¹, Mudassar Mohiuddin^1*, Ayesha Mohiud Din^2,3, Tahira Kamal⁴, Muhammad Khalid Mansoor¹,

Abubakar Siddique^5,6,7, Mudasser Habib⁸, Riaz Hussain⁹, Muhammad Taslim Ghori¹⁰, Hira Hameed¹, Humaira Rizwana¹¹,

Islem Abid¹², Muhammad Ehsan¹³, Aroosh Shabbir¹⁴

¹Department of Microbiology, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Pakistan

²Department of Biotechnology, Virtual University of Pakistan, Lahore, Pakistan

³School of Biological Sciences, University of Southampton, Southampton, UK

⁴National Institute for Genomics and Advanced Biotechnology, National Agricultural Research Centre (NARC), Islamabad, Pakistan

⁵Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Science and Technology (NUST), Islamabad, Pakistan

⁶Department of Veterinary Medicine, College of Animal Sciences, Zhejiang University, China

⁷Hainan Institute of Zhejiang University, Sanya, China

⁸Animal Sciences Division, Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan

⁹Department of Pathology, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Pakistan

¹⁰Department of Clinical Medicine and Surgery, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Pakistan

¹¹Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia

¹²Centre of Excellence in Biotechnology Research, King Saud University, P.O Box 2455, Riyad 11495, Saudi Arabia

¹³Department of Parasitology, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Pakistan

¹⁴Institute of Molecular Biology and Biotechnology (IMBB), The University of Lahore, Pakistan

Abstract

The lumpy skin disease virus (LSDV) is a member of the Capripox genus of the Poxviridae family. It is the causative agent of lumpy skin, a highly contagious disease of cattle, water buffalo, sheep, and goats. In 2022, several outbreaks of LSD were reported in the Cholistan region of Pakistan, which has a large population of livestock living in arid conditions. A total of 230 blood/serum and scab samples were collected from three LSD outbreak locations. Clinically, affected cattle showed acute clinical signs characterized by skin nodules, fever, enlarged lymph nodes, emaciation, and lower leg edema. Hematological findings revealed non-significant changes in red blood cell and white blood cell counts (some animals had leukocytosis while others were leukopenic) whereas, hemoglobin level were significantly low. Platelet count, MPV, PCT, P-LRC, and P-LCC were elevated. Granulocytes were significantly low in LSD affected cattle while lymphocyte counts were significantly high. Serological findings revealed elevated protein levels, along with high creatinine and ALT concentrations. Amplification of DNA-dependent RNA polymerase 30 kDa subunit gene (RPO30) confirmed the presence of LSD virus in all suspected samples. Phylogenetic analysis showed that all Pakistani isolates clustered closely with isolates from neighboring countries. The SNPs differences were less than 20 among these isolates, indicating their close resemblance with each other. It can, therefore, be inferred that our LSD strains might be originated from neighboring Asian countries, that were affected by LSD in previous years.

Keywords: Lumpy skin disease virus (LSDV), Cholistan, Skin lesions, Hematology, Phylogenetic analysis

Pozdnyakova Yelena^1*, Solyanov Dmitry², Tatina Yelena¹, Britko Valeriy¹, Omarbekova Nazgul³, Korshukova Marina³

¹Department of Biomedicine, Karaganda Medical University, Karaganda, The Republic of Kazakhstan

²Department of Pharmacy, Karaganda Medical University, Karaganda, The Republic of Kazakhstan

³Department of Informatics and Biostatistics, Karaganda Medical University, Karaganda, The Republic of Kazakhstan

Abstract

The aim of this study is to investigate the effects on the nervous system of the use of Stachys sieboldii root powder, in growing male rats on a high fat and sucrose (HFHS) diet. The animals were divided into three groups: Intact – normal rodent chow, HFHS – high fat and sucrose diet, HFHS + Stachys – high fat and sucrose diet with Stachys supplementation. After 30 days of the experiment, the animals were subjected to the “Open Field” test to study the changes in behavioural responses. Biochemical studies of changes in the concentration of lipid peroxidation products in brain homogenates of growing rats were also carried out. Growing males on the HFHS diet exhibit altered nervous system functioning. Animals show apathy, their motor activity and indicators of exploratory behaviour are reduced and the level of emotional reactivity increases. On the part of biochemical changes – the level of all indicators of lipid peroxidation increases significantly. The introduction of Stachys root powder reduces the negative effect of the diet on the organism of animals – they significantly increase the indicators of exploratory behaviour, increase locomotor activity and reduce emotional reactivity, as well as there is a decrease in the concentration of all products of free-radical oxidation in the brain homogenate. Our study showed that diets high in fat and sucrose had negative effects on growing male rats. The introduction of Stachys sieboldii root powder into the diet improved both behavioural response scores and antioxidant processes in the brain.

Keywords: Stachys sieboldii, Open field, Lipid peroxidation, Conjugated dienes, Ketodienes

Maria Rasool, Muhammad Hidayat Rasool^*, Mohsin Khurshid, Bilal Aslam

Institute of Microbiology, Government College University Faisalabad, Faisalabad-38000, Pakistan

Abstract

Multi-drug resistant (MDR) bacterial infections significantly increase mortality, morbidity, and treatment costs when they persist. Therefore, there is a pressing need to discover, modify, or search for antimicrobial agents with the ability to combat MDR bacteria. Silver nanoparticles used in this study were synthesized by Bacillus subtilis and characterized through different techniques. MDR strains underwent antibacterial activity, antioxidant activity, and time-kill kinetic assays to assess susceptibility to silver nanoparticles. Furthermore, the synergistic impact of silver nanoparticles and antibiotics was examined using the two-dimensional checkerboard method to calculate the Fractional Inhibitory Concentration Index (FICI). Scanning Electron Microscopy (SEM) results revealed a circular shape of synthesized AgNPs, with an average length and area of 76.78 and 47.10 nm, respectively. UV analysis showed an optimum peak at 420 nm. XRD analysis indicated the crystalline nature of nanoparticles with diversity in size. Remarkable antioxidant potential (55% of AgNPs) was observed at a concentration of 1000 µg/ml, while minimum activity (18%) was noted at 62.6 µg/ml. Silver nanoparticles demonstrated a synergistic interaction with the antibiotic Cefixime against Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae, with FICI values of 0.37, 0.3, 0.25, and 0.49, respectively, and an additive effect against Pseudomonas aeruginosa, with a FICI value of 0.7. Moreover, this research explores the anti-inflammatory potential of silver nanoparticles (AgNPs) in two distinct models: formaldehyde-induced inflammation and carrageenan-induced inflammation, along with an assessment of their in vitro anti-inflammatory activity. The findings shed light on the multifaceted role of AgNPs in mitigating inflammatory responses, offering promising avenues for therapeutic interventions.

Keywords: Green synthesis, Silver, Bacillus subtilis, Synergistic effect, Cefixime, XRD

Urairat Mongmonsin¹, Prapenpuksiri Rungsa², Withan Teajaroen¹, Theerasak Somdee³, Jureerut Daduang⁴, Sakda Daduang^2,5*

¹Department of Biomedical Sciences, Faculty of Graduate School, Khon Kaen University, Khon Kaen, Thailand

²Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen, Thailand

³Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand

⁴Department of Clinical Chemistry, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand

⁵Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand

Abstract

Cordyceps militaris has been used in traditional medicine due to its immune-boosting functions, especially in elderly and cancer patients. The study aims to assess the impact of C. militaris extract on the immune response in LPS-induced splenocytes and male Sprague Dawley rats. The study found that CME contains various bioactive compounds such as adenosine, cordycepin (3′ deoxyadenosine), and phenolic compounds. In vitro studies showed that CME has the potential to enhance CD3⁺ T cell and CD45RA⁺ B cell populations in LPS-induced splenocytes with a cell viability of over 80%. Moreover, we found that CME can enhance the immune response in rats by increasing CD3⁺ T cell proliferation and up-regulating IL-4, IL-6, and IL-17a expression, without affecting the rat’s body weight. The findings suggest that CME could be a promising immune enhancer for patients with weakened immune systems. However, further animal experiments with varying doses are essential to determine the optimal dosage for the successful development of CME as a therapeutic agent.

Keywords: Cordyceps militaris, Immunostimulatory effects, Pro-inflammatory cytokines, Cordycepin