Serum and salivary Cu/Zn ratio as a diagnostic biomarker for oral submucosal fibrosis: an analysis of trace metals and LOX gene variants.

This study aimed to analyze the serum and salivary levels of copper (Cu), zinc (Zn), iron (Fe), chromium (Cr), manganese (Mn) and the Cu/Zn ratio and investigate the association between LOX gene variants (rs18800449 and rs2288393) and oral submucosal fibrosis (OSMF). A total of 250 subjects were included in the study: OSMF patients (n = 50), areca nut chewers without OSMF (n = 100) and controls (n = 100). Trace metals were measured using an atomic absorption spectrophotometer, while LOX gene variants were genotyped using the tetra primer amplification refractory mutation system (tetra ARMS) polymerase chain reaction (PCR) method. The results showed significant variations in serum and salivary Cu, Zn, Fe and Cr levels and serum Mn concentrations among the three groups (p < 0.0001). Serum Cu levels were significantly higher in OSMF patients, while serum Zn levels were significantly lower. Both serum and salivary Cu/Zn ratios demonstrated a statistically significant difference (p < 0.0001) and diagnostic potential to differentiate OSMF from chewers and controls. However, LOX gene variants did not show an association between OSMF and chewers, except for rs1800449 genotypes, which showed a significant and increased risk with the AA genotype in OSMF patients compared to controls (OR = 7.58; 95%CI 2.30-24.97). The study suggests that trace elements and genetic variants may impact the etiology of OSMF. The findings may aid in early diagnosis, suitable treatment, and as a prognostic indicator for disease progression.

Spectrum Of HBB Gene Variants And Major Endocrine Complications In Thalassemia Patients Of Pakistan.

OBJECTIVE: To determine the molecular characterisation of beta-thalassemia major patients, pattern of major endocrine complications and its association with haemoglobin subunit beta gene variants. Method: The cross-sectional study was conducted from November 2021 to November 2022 after approval from the ethics review committee of Liaquat University of Medical and Health Sciences, Jamshoro, Pakistan, and comprised of 88 patients with beta thalassemia major aged >8 years and having serum ferritin level >1000 µg/L. The subjects were analysed for haemoglobin subunit beta gene variants and major endocrine complications, like growth retardation, hypogonadism, hypothyroidism, hypoparathyroidism and diabetic abnormalities using an automatic chemistry analyser, fully automatic chemiluminescence immunoassay analyser, enzyme-linked immunosorbent assay and real-time polymerase chain reaction. Data was analysed using SPSS 25. RESULTS: Of the 88 subjects, 40(45.4%) were girls and 48(54.5%) were boys. The overall mean age was 12±2.81 years. Of the total, 55(62.5%) had growth retardation, 41(46.6%) were cases of hypogonadism, 16(18.1%) hypothyroidism, 5(5.7%) hypoparathyroidism, 3(3.4%) diabetes mellitus and 8 (9.1%) had impaired glucose tolerance. Also, 65 (73.9%) patients confronted at least one endocrine complication. Endocrine complications were strongly associated with serum ferritin levels (p=0.000). The most common haemoglobin subunit beta gene variant identified was IVSI-5 (G>C) in 36 (40.9%), and the least identified variant was cluster of differenctiation-CD26(G>A) 1(1.1%). The association between haemoglobin subunit beta gene variants with endocrine complications was statistically non-significant (p>0.05). CONCLUSIONS: IVSI-5 (G>C) was found to be the most frequent haemoglobin subunit beta gene variant among beta- thalassemia major patients.

Factors And Determinants Associated With Prevalence Of Anaemia In Adults In Karachi, Pakistan.

Objectives: To assess the demographic and dietary factors associated with the prevalence of anaemia in multi-ethnic urban settings. METHODS: The cross-sectional community-based survey study was conducted from August 2020 to May 2021 in Karachi East district, and comprised healthy adults of either gender aged 20-60 years. Data was collected using a pretested questionnaire. Besides, 4ml sample of whole blood was taken from each participant for haematological analysis. Data was analysed using SPSS 23. RESULTS: Of the 416 subjects, 269(64.7%) were males and 147(35.3%) were females, while 334(80.3%) were aged <30 years and 82(19.7%) were aged >30 years. Anaemia was found in 92(22.1%) subjects. Female gender, lower and middle socioeconomic class, nuclear family type, habit of meal-skipping, and infrequent consumption of sweets and milk were all linked to anaemia (p<0.05). CONCLUSIONS: Less than a quarter of the sample was found to be anaemic. Steps should be taken to address the identified causes of anaemia.

Association of Equiliberative Nucleoside Transporter (ENT) with liver fibrosis stage in Hepatitis-C.

OBJECTIVE: To evaluate the pattern of equiliberative nucleoside transporters in viral hepatitis responding and non-responding patients, to correlate the liver fibrosis stage with the pattern among the non-responders, and to correlate the equiliberative nucleoside transporter status with housekeeping hypoxanthine-guanine phosphoribosyltransferase gene. Method: The comparative cross-sectional study was conducted at the Molecular Biology and Genetics Department, Liaquat University of Medical and Health Sciences, Jamshoro, Pakistan, from March to August 2018, and comprised adult hepatitis C virus patients of either gender who completed six months of treatment. They were assessed for response to therapy in terms of the presence of the viral load in their serum by using real time polymerase chain reaction, and divided into responder group A and non-responder group B. The groups were compared and correlation between equiliberative nucleoside transporter expressions and liver fibrosis was evaluated. Data was analysed using SPSS 23. RESULTS: Of the 80 patients, 33(41.3%) were males and 47(58.8%) were females. The overall mean age was 37.46±10.61 years. In terms of response to treatment, there were 40(50%) in each of the two groups. Mean post-treatment duration was 15.38±30.09 weeks. Age was not significantly different with respect to gender (p>0.05), but the age pattern was significantly different between the two groups (p<0.001). Also, non-responders had significant post-treatment duration compared to the responders (p<0.001). Hypoxanthine-guanine phosphoribosyltransferase gene showed no significant difference between the groups (p=0.144). Equiliberative nucleoside transporter was significantly down-regulated in the non-responders (p<0.001) and showed correlation with the degree of liver fibrosis (p<0.034) compared to the responders. CONCLUSIONS: There was a significant association between equiliberative nucleoside transporters and liver fibrosis stage in hepatitis C virus non-responding patients.

Tumour infiltrating lymphocytes in colorectal cancers- correlation with tumour biology and clinical outcome: A cohort study.

OBJECTIVE: To evaluate the pattern of tumour infiltrating lymphocytes in colorectal cancers, and to correlate them with nuclear protein Ki67, vascular endothelial growth factor and clinical outcome. METHODS: The retrospective study was conducted at the Nuclear Institute of Medicine and Radiotherapy and the Liaquat University of Medical and Health Sciences, Jamshoro, Pakistan, and comprised data of colorectal cancer patients from January 1, 2008, to December 31, 2018. Whole tumour sections of colorectal cancer were used with haematoxylin and eosin staining, Histological type, grade and infiltrated lymphocytes within the tumour block were assessed. Ki67 and vascular endothelial growth factor were evaluated by immunohistochemistry, while the staining of these biomarkers was assessed by the percentage of cells stained. Data was analysed using SPSS 22. RESULTS: Of the 201 patients, 110(54.7%) were males and 91(45.3%) were females. Overall median age was 43 years (range 10-85 years). Majority of the tumours 132(65.7%) showed mild to moderate tumour infiltrating lymphocytes, 30(14.9%) had severe tumour infiltrating lymphocytes, while 39(19.4%) did not show any infiltrating lymphocytes. Tumour infiltrating lymphocytes did not show significant association with the histological grade (p>0.05), but high tumour infiltrating lymphocytes were associated with poor survival without being significantly associated with Ki67 pattern and vascular endothelial growth factor (p>0.05). CONCLUSIONS: Majority of colorectal cancer cases showed varying levels of lymphocyte infilteration, and tumour infiltrating lymphocytes were associated with poor survival, without having significant association with Ki67 pattern and vascular endothelial growth factor.

The Dawn of next generation DNA sequencing in myelodysplastic syndromes- experience from Pakistan.

BACKGROUND: Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells exhibiting ineffective hematopoiesis and tendency for transformation into acute myeloid leukemia (AML). The available karyotyping and fluorescent in situ hybridization provide limited information on molecular abnormalities for diagnosis/prognosis of MDS. Next generation DNA sequencing (NGS), providing deep insights into molecular mechanisms being involved in pathophysiology, was employed to study MDS in Pakistani cohort. PATIENTS AND METHODS: It was a descriptive cross-sectional study carried out at National institute of blood diseases and bone marrow transplant from 2016 to 2019. Total of 22 cases of MDS were included. Complete blood counts, bone marrow assessment and cytogenetic analysis was done. Patients were classified according to revised WHO classification 2016 and IPSS score was applied for risk stratification. Baseline blood samples were subjected to analysis by NGS using a panel of 54 genes associated with myeloid malignancies. RESULTS: The median age of patients was 48.5 ± 9.19 years. The most common presenting complaint was weakness 10(45.45%). Cytogenetics analysis revealed abnormal karyotype in 10 (45.45%) patients. On NGS, 54 non-silent rare frequency somatic mutational events in 29 genes were observed (average of 3.82 (SD ± 2.08) mutations per patient), including mutations previously not observed in MDS or AML. Notably, two genes of cohesin complex, RAD21 and STAG2, and two tumor suppressor genes, CDKN2A and TP53, contained highest number of recurrent non-silent somatic mutations in the MDS. Strikingly, a missense somatic mutation p.M272Rof Rad21 was observed in 13 cases. Overall, non-silent somatic mutations in these four genes were observed in 21 of the 22 cases. The filtration with PharmGKB database highlighted a non-synonymous genetic variant rs1042522 [G > C] located in the TP53. Genotype GG and GC of this variant are associated with decreased response to cisplatin and paclitaxel chemotherapy. These two genotypes were found in 13 cases. CONCLUSION: Sequencing studies suggest that numerous genetic variants are involved in the initiation of MDS and in the development of AML. In countries like Pakistan where financial reservation of patients makes the use of such analysis even more difficult when the availability of advanced techniques is already a prevailing issue, our study could be an initiating effort in adding important information to the local data. Further studies and large sample size are needed in future to enlighten molecular profiling and ultimately would be helpful to compare and contrast the molecular characteristics of Asian versus global population.

The clinical and demographical profile of Coronavirus illness: The tale of Tablighi Jamaat and Zaireen in Quarantine / Isolation center at Sukkur and Hyderabad.

OBJECTIVES: To determine the clinical and demographical profile of corona-virus illness among Tablighi Jamaat and Zaireen kept in quarantine / isolation center at Sukkur and Hyderabad Sindh. METHODS: The cross-sectional descriptive study (late March-2020 to mid of April-2020) was conducted at Diagnostic & Research Laboratory LUMHS Jamshoro / Hyderabad. All the suspected cases for COVID-19 were recruited and screened for corona virus infection. The study explored the data of the suspected and diagnosed (confirmed) case of COVID-2019 (Tablighi Jamaat and Zaireen) reported by Diagnostic Research Laboratory Liaquat University of Medical and Health Sciences (LUMHS) Jamshoro who belonged to various parts of the country in general and province Sindh in particular. All the individuals regardless of age and gender presented either as asymptomatic, critical ill or having non-specific symptoms as fever, flu, cough; sore throat and shortness of breath were screened for COVID-19 by real time PCR after taking informed consent whereas the frequency / percentages (%) and means ±SD computed for study variables. RESULTS: During study period total 920 patients were explored and screened for Corona virus infection. The mean ± SD for age (yrs) of overall population of city Sukkur and Hyderabad was 57.83±8.84 and 59.62±9.72 respectively. The 700 people from Sukkur city was screened and out of them 276 (39.4%) were positive and 424 (60.5) were negative while the cure rate was 245 (88.7%) along with mean ± SD for recovery time was 9.41±2.97. The 220 people from Hyderabad city was screened and out of them 106 (48.1%) were positive and 114 (51.8%) were negative while the cure rate was 106 (100%) along with mean ± SD for recovery time was 11.54±3.42. The majority of cases at both centers were asymptomatic (90%), symptomatic (7%) and critically ill (3%). The mortality accounted for 2.8% cases at Hyderabad isolation center and all were having smoking history and co-morbidities as ischemic heart diseases, diabetes mellitus, obstructive lung disease and cerebrovascular accident whereas no mortality was observed at Sukkur isolation center. CONCLUSION: RT-PCR measure allowed fast, delicate, and explicit discovery of SARS-CoV in biochemical diagnosis. The majority of cases at both centers were asymptomatic while the mortality was identified in 2.8% cases (having co-morbidities) at Hyderabad isolation center whereas no mortality was observed at Sukkur isolation center.

MTHFR and F5 genetic variations have association with preeclampsia in Pakistani patients: a case control study.

BACKGROUND: To study the role of single nucleotide variants (SNVs) of genes related to preeclampsia in Pakistani pregnant women. METHODS: After ethical approval and getting informed consent; 250 pregnant women were enrolled and equally divided into two groups (125 preeclamptic cases and 125 normotensive pregnant women). Demographic details and medical history were recorded, and 10 ml blood sample was obtained for DNA extraction. The tetra-primer amplification refractory mutation system (ARMS) assays were developed for assessing the variants of three preeclampsia related genes; F5, MTHFR and VEGFA. An association of six SNVs; F5:c.1601G > A (rs6025), F5:c.6665A > G (rs6027), MTHFR: c.665C > T (rs1801133), MTHFR: c.1286A > C (rs1801131), VEGFA: c.-2055A > C (rs699947) and VEGFA: c.*237C > T (rs3025039) with preeclampsia was determined by using different genetic models. RESULTS: Genotyping of the SNVs revealed that patients with MTHFR:c.665C > T, have increased susceptibility to preeclampsia (CT versus CC/TT: OR = 2.79, 95% CI = 1.18-6.59; P* = 0.046 and CT/TT vs CC: OR = 2.91, 95% CI = 1.29-6.57; P* = 0.0497, in overdominant and dominant models, respectively), whereas F5:c.6665A > G, (A/G vs AA/GG: OR = 0.42, 95% CI = 0.21-0.84; P* = 0.038 in overdominant model) and MTHFR:c.1286A > C, (CC versus AA: OR = 0.36, 95% CI = 0.18-0.72; P* = 0.0392 in codominant model) have significantly decreased risk for preeclampsia. F5:c.1601G > A, VEGFA: c.-2055A > C and VEGFA: c.*237C > T variants revealed no relationship with the disease. CONCLUSION: This is the first case control study describing the protective role of F5:c.6665A > G against preeclampsia in any world population. In addition, the present study confirmed the association and role of MTHFR gene variations in the development of preeclampsia in Pakistani patients. Further genetic studies may be required to better understand the complex genetic mechanism of SNVs in preeclampsia related genes in pregnant women.

Correction to: Identification of novel mutations in congenital afibrinogenemia patients and molecular modeling of missense mutations in Pakistani population.

[This corrects the article DOI: 10.1186/s12959-017-0143-3.].

HIV-1 genetic diversity, geographical linkages and antiretroviral drug resistance among individuals from Pakistan.

Worldwide antiretroviral therapy (ART) has reduced the mortality and morbidity rates in individuals with HIV infection. However, the increasing occurrence of drug resistance is limiting treatment options. In recent years, Pakistan has witnessed a concentrated epidemic of HIV. It is very important to identify geographical linkages and mutations that generate selective pressure and drive resistance of HIV in our population. The aim of this work was to identify genetic diversity and drug resistance patterns of HIV in Pakistan, using available sequences and bioinformatics tools, which may help in selecting effective combination of available drugs. A total of 755 Pakistani HIV gag, pol and env sequences were retrieved from the Los Alamos HIV database. Sequences were aligned with reference sequences of different subtypes. For geographical linkages, sequences of predominant subtypes were aligned with sequences of the same subtypes from different countries. Phylogenetic trees were constructed using the maximum-likelihood method in MEGA 7 software. For drug resistance analysis, sequences were entered into the Stanford University HIV Drug Resistance Database. Phylogenetic trees for studying genetic diversity showed that 82% of the sequences were of subtype A, while the rest of the sequences were of subtypes B (9.5%), K (2%), D (2%) and AE (1%). Moreover, trees that were constructed to examine geographical linkages showed close clustering of strains with those of the neighboring countries Afghanistan and India, as well as some African countries. A search for drug resistance mutations showed that 93% of the sequences had no major or minor mutations. The remaining 7% of the sequences contained a major mutation, Y115F, which causes the virus to exhibit low to intermediate resistance against lamivudine and emtricitabine. Our data indicate that HIV subtype A is the major subtype, while subtypes K, D and AE are also present in our country, suggesting gradual viral evolution and possible entry of different subtypes from neighboring countries. These data suggest that HIV is still sensitive to most of the antiretroviral drugs used in Pakistan.

Correction to: HIV-1 genetic diversity, geographical linkages and antiretroviral drug resistance among individuals from Pakistan.

Unfortunately, the co-author's name Nouman Mughal was incorrectly published in the original article and the same is corrected here in this erratum.

Identification of novel mutations in congenital afibrinogenemia patients and molecular modeling of missense mutations in Pakistani population.

BACKGROUND: Congenital afibrinogenemia (OMIM #202400) is a rare coagulation disorder that was first described in 1920. It is transmitted as an autosomal recessive trait that is characterized by absent levels of fibrinogen (factor I) in plasma. Consanguinity in Pakistan and its neighboring countries has resulted in a higher number of cases of congenital fibrinogen deficiency in their respective populations. This study focused on the detection of mutations in fibrinogen genes using DNA sequencing and molecular modeling of missense mutations in all three genes [Fibrinogen gene alpha (FGA), beta (FGB) and gamma (FGG)] in Pakistani patients. METHODS: This descriptive and cross sectional study was conducted in Karachi and Lahore and fully complied with the Declaration of Helsinki. Patients with fibrinogen deficiency were screened for mutations in the Fibrinogen alpha (FGA), beta (FGB) and gamma (FGG) genes by direct sequencing. Molecular modeling was performed to predict the putative structure functional impact of the missense mutations identified in this study. RESULTS: Ten patients had mutations in FGA followed by three mutations in FGB and three mutations in FGG, respectively. Twelve of these mutations were novel. The missense mutations were predicted to result in a loss of stability because they break ordered regions and cause clashes in the hydrophobic core of the protein. CONCLUSIONS: Congenital afibrinogenemia is a rapidly growing problem in regions where consanguinity is frequently practiced. This study illustrates that mutations in FGA are relatively more common in Pakistani patients and molecular modeling of the missense mutations has shown damaging protein structures which has profounding effect on phenotypic bleeding manifestations in these patients.

Seroprevalence of transfusion transmitted infections among different blood group donors at Blood Bank LUMHS, Hyderabad.

OBJECTIVES: To study the prevalence of HBsAg, Anti-HCV, HIV, Syphilis and Malaria in blood donors. METHODS: This is a cross sectional descriptive study, conducted at Blood bank and Transfusion center at Liaquat University of Medical & Health Sciences (LUMHS) Hyderabad, during the period from January, 2014 to June, 2015. A total of 4683 blood donors were screened for HBsAg, Anti-HCV and HIV on Architect 20001 (manufactured by Abbott), employing chemiluminescent microparticle immunoassay (CMIA). For Syphilis, VDRL ICT kits were used and Malaria parasite was screen through MP slides. Blood grouping was performed by both forward and reverse methods. RESULTS: This study showed a high frequency of HBsAg, VDRL and malaria positivity among the O-ve blood group donors, i.e. 3.70%, 9.25% and 0.61% respectively. Blood group B-ve individuals were commonly infected with HCV (12.5%) as compared with all other blood group donors. HIV is more commonly reported in A+ve blood group individuals. Blood group O+ve is more prevalent (37.41 %). CONCLUSION: High frequency of HCV infection in blood donors advocates implementation of strict screening policy for donors and public awareness campaigns about preventive measures to reduce the spread of this infection as well as other transfusion transmissible infections.

Cytogenetic and Molecular Analyses of Philadelphia Chromosome Variants in CML (chronic myeloid leukemia) Patients from Sindh using Karyotyping and RT-PCR.

OBJECTIVE: To determine the frequency of Philadelphia chromosome (Ph) and its variants in chronic myeloid leukemia (CML) cases at a tertiary care hospital of Sindh. METHODS: The study was conducted at the Department of Pathology, Liaquat University of Medical and Health Sciences, Jamshoro and Isra University Hospital, Hyderabad during May-to-September 2014. Bone marrow and peripheral blood samples from a total of 145 diagnosed cases of CML were collected. Cytogenetic analyses were performed using karyotyping as per the International System for Human Cytogenetic Nomenclature guidelines. All karyotypic images were analyzed using the Cytovision software. In order to identify BCR-ABL transcripts, RT-PCR was performed. Statistical analysis of the data was done using SPSS-version-21.0. RESULTS: Of the 145 samples, a total of 133 (91.7%) were positive for the Ph (Ph+) while 12 (8.3%) were negative for the Ph (Ph-). Of the 133 Ph+ samples, standard karyotypes were noted in 121 (91%), simple variants in 9 (6.7%) and complex variants in 3 (2.3%) of the samples. All the Ph+ samples (n=133) showed BCR-ABL positivity. Of the 12 Ph- samples, a total of 7 (58.3%) were BCR-ABL-positive and 5 (41.6%) were BCR-ABL-negative. CONCLUSION: Frequency of the Ph was found to be of 90.9% in CML patients using a highly sensitive technique, the RT-PCR. Cytogenetic abnormalities were at a lower frequency. Cytogenetic and molecular studies must be conducted for better management of CML cases. These findings could be very useful in guiding the appropriate therapeutic options for CML patients.

Relationship between aging and control of metabolic syndrome with telomere shortening: a cross-sectional study.

Aging is considered one of the major risk factors for several human disorders. The telomere plays a crucial role in regulating cellular responsiveness to stress and growth stimuli as well as maintaining the integrity of the Deoxyribonucleic Acid (DNA), and aging leads to the progressive decline in the telomere length (TL) due to continuous cell division. The aim of this study was to determine the relationship between TL and advancing age and the impact of metabolic syndrome (MetS) on TL. Firstly, we determined the association of advancing age and TL, by measuring telomere length (T/S ratio) in healthy volunteers (n = 90). The TL was compared between normal population and patients with metabolic syndrome (n = 298). The age matched controlled and uncontrolled MetS patients (n = 149) were also compared for their TL T/S ratio. The TL showed negative correlation with advancing age, whereas the significant change was observed at the cut-offs of 40 and 70 years defining 40 with longer TL and 70 as shorter TL. The longest T/S ratio at 2.46 was measured at the age range of 1 year in healthy volunteers, while elderly population showed considerably shorter TL. The patients older than 60 years with poor or uncontrolled MetS had shorter TL, as compared to the controlled MetS. In conclusion our findings suggest that TL was negatively correlated with advancing age. Uncontrolled metabolic syndrome appeared to have worsening effects on TL. Telomere length appears to have potential to be used a parameter to determine age. However, further large scale studies are recommended to make firm guidelines.

Impact of ACE2 gene variations on COVID-19 pathogenicity in Pakistani patients.

Background: COVID-19, caused by the SARS-CoV-2 virus, swiftly disseminated and was declared a pandemic. Variations in the ACE2 gene can impact the virus's ability to bind to ACE2 receptor, potentially influencing an individual's susceptibility and its association with COVID-19 severity across various populations. Methods: In total, 200 individuals were sequenced for the ACE2 gene and potential impact of the found variants on the ACE2 protein was assessed using in-silico tools. Results: Eight variations in the ACE2 gene were identified in 27 COVID-19 patients, of which four were missense and four were intronic variants. Three variants had a MAF of < 0.01 (c.251C > T, p.Pro86Leu; 15C > G, p.S5S; and c. 91 A > G, p.Lys31Glu). A missense variant, p.Pro86Leu, C > T, TT genotype, was found in 9 out of 200 individuals with an allele frequency of 0.045 and showed a significant association with COVID-19 (P = 0.003). The heterozygous allele of 15C > G, p.S5S, was found with a frequency of 0.02 (8/400) in eight patients, and its CG genotype showed a significant association with COVID-19 (P = 0.0068). The remaining identified variants were not associated with COVID-19 susceptibility. Conclusion: The ACE2 gene sequence in Pakistani individuals exhibited a low frequency of identified variants in COVID-19 patients. Overall, only two variants were associated with susceptibility to the disease, possibly contributing to Pakistan's lower COVID-19 mortality and infection rates. However, individuals carrying the mutant variant experienced more severe symptoms.

The possible "calming effect" of subchronic supplementation of a standardised phospholipid carrier-based Melissa officinalis L. extract in healthy adults with emotional distress and poor sleep conditions: results from a prospective, randomised, double-blinded, placebo-controlled clinical trial.

Background: Emotional distress conditions such as depression, anxiety, stress, and poor sleep are widespread health problems that have a significant impact on people's lives. Conventional drugs are commonly prescribed to treat emotional distress and poor sleep conditions; however, these medications have several limitations and have shown multiple side effects. Over recent years botanicals-based pharmacological agents have gained increasing research and clinical interest in the management of emotional distress and sleep disorder. Of note, Melissa officinalis L. (MO) leaf extract has demonstrated considerable neuropharmacological properties both in animal and human studies and has emerged as a promising natural "calming agent." However, research in this area is limited, and more studies are needed to validate its efficacy in amelioration of emotional distress and poor sleep conditions. Objectives: We aimed to assess the pharmacological effects of subchronic supplementation of an innovative standardised phospholipid carrier-based MO aqueous extract on emotional distress and poor sleep conditions. Design: A 3-week prospective, randomised, placebo-controlled, parallel-group, double-blinded clinical trial was conducted in 100 healthy adults complaining of a moderate degree of depression, anxiety, or stress, with scores of ≥14, ≥10, and ≥19, respectively, in the self-report Depression, Anxiety, and Stress Scale (DASS-42) or poor sleep, as indicated by the score of >5 in the Pittsburgh Sleep Quality Index (PSQI) scale. In addition, the impact of emotional distress and/or poor sleep on participants' mental wellbeing, emotional feelings, and quality of life was also assessed using the self-reported Warwick-Edinburgh Mental Wellbeing Scale (WEMWBS), Positive and Negative Affect Schedule (PANAS) scale, and quality of life (WHO-QoL-BREF) scale, respectively. Results: Oral supplementation of 200 mg of phospholipid-based MO aqueous extract (Relissa™) tablets twice a day (i.e., 400 mg/day) for 3 weeks led to significant improvements in the depressive mood, anxiety, stress, positive and negative affect (emotional feelings), overall mental wellbeing, and quality-of-life scores (all p values <0.001). Supplementation of MO extract was well tolerated, and no treatment-emergent effects or serious adverse events were reported. Conclusion: According to the results of this study, the phospholipid carrier-based MO aqueous extract possesses considerable neuropharmacological properties, and its supplementation may provide a promising therapeutic option for the management of moderate emotional distress and/or poor sleep conditions. Clinical Trial Registration: clinicaltrials.gov, identifier NCT05602688.

Delineating the Spectrum of Genetic Variants Associated with Bardet-Biedl Syndrome in Consanguineous Pakistani Pedigrees.

This study aimed to find the molecular basis of Bardet-Biedl syndrome (BBS) in Pakistani consanguineous families. A total of 12 affected families were enrolled. Clinical investigations were performed to access the BBS-associated phenotypes. Whole exome sequencing was conducted on one affected individual from each family. The computational functional analysis predicted the variants' pathogenic effects and modeled the mutated proteins. Whole-exome sequencing revealed 9 pathogenic variants in six genes associated with BBS in 12 families. The BBS6/MKS was the most common BBS causative gene identified in five families (5/12, 41.6%), with one novel (c.1226G>A, p.Gly409Glu) and two reported variants. c.774G>A, Thr259LeuTer21 was the most frequent BBS6/MMKS allele in three families 3/5 (60%). Two variants, c.223C>T, p.Arg75Ter and a novel, c. 252delA, p.Lys85STer39 were detected in the BBS9 gene. A novel 8bp deletion c.387_394delAAATAAAA, p. Asn130GlyfsTer3 was found in BBS3 gene. Three known variants were detected in the BBS1, BBS2, and BBS7 genes. Identification of novel likely pathogenic variants in three genes reaffirms the allelic and genetic heterogeneity of BBS in Pakistani patients. The clinical differences among patients carrying the same pathogenic variant may be due to other factors influencing the phenotype, including variants in other modifier genes.

The possible therapeutic role of curcumin and quercetin in the early-stage of COVID-19-Results from a pragmatic randomized clinical trial.

Background: Curcumin (CUR) and quercetin (QUE), two natural polyphenols, possess diverse biological activities including broad-spectrum antiviral, antioxidant, and immunomodulatory effects. Both CUR and QUE have shown inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in in vitro assays. Objective: In the present study we aimed to assess the possible treatment benefits of a combined curcumin and quercetin (CUR-QUE) oral supplement, alongside standard of care (SOC), in the early-stage COVID-19 infection. Methods: This was an exploratory, pragmatic, open-label, randomized controlled clinical trial, conducted at the Department of Pathology, Liaquat University of Medical and Health Sciences, Jamshoro, PK. The study compared the treatment effect of an oral CUR-QUE supplement plus SOC vs. SOC alone, in the early-stage/mild to moderately symptomatic COVID-19 outpatients. Patients were randomized in a 1:1 ratio to CUR-QUE (n = 25) and control (n = 25) treatment groups. The CUR-QUE supplementation consisted of a daily intake of 168 mg curcumin and 260 mg quercetin, as two soft capsules, to be taken twice a day at home for 14 days. Results: After one-week of treatment, most of the patients in the CUR-QUE group showed an expedited clearance of the viral infection i.e., 18 (72.0%) vs. 6 (24.0%) patients in the control group tested negative for SARS-CoV-2 in the nasal-oropharyngeal swab reverse transcription-polymerase chain reaction (RT-PCR) analysis (p = 0.0002). In addition, COVID-19-associated acute symptoms were also speedily resolved in the CUR-QUE treated patients, i.e., 10 (40.0%) vs. 4 (16.0%) patients in the control group (p = 0.061). The CUR-QUE supplementation therapy was well-tolerated by all 25 patients and no treatment-emergent effects or serious adverse events were reported. Conclusion: The results revealed in this exploratory study suggest a possible therapeutic role of curcumin and quercetin in the early-stage of COVID-19. It is proposed that the two agents possibly acting in synergy, interfere the SARS-CoV-2 replication, and thus help a speedy recovery in the early-stage of COVID-19. Further research is highly encouraged. Clinical trial registration: Clinicaltrials.gov, Identifier NCT04603690.

Novel molecular classification of colorectal cancer and correlation with survival.

Introduction: Colorectal cancer (CRC) is one of the most common cancers worldwide. This study was designed to evaluate biological patterns, explore molecular classification and correlate with survival outcome in treatment naïve CRC patients. Methods: Over 11 years consecutive series of 435 CRC patients were operated on as primary surgical therapy. A total of 201 CRC patients were included, whose complete set of clinical information was available, and their good quality tumour blocks were retrieved. Immunohistochemistry was used for tumour analysis, and partitional clustering was performed using R software for cluster analysis. Results: The median age was 43 (range 10-85) years; adenocarcinoma was the most commonly seen histological type. The great majority had positive CK20, CEA, E-Cadherin, Ki67, CDX2, and p53 expression. There were four distinct molecular classes found, whereas Ki67, CDX2, and p53 play the main role in partitioning. Younger age negatively impacted survival; overall and disease-specific survival was 26 months only with 50 months' longest survival. Conclusion: Colorectal cancer is a biologically heterogeneous disease with at least four distinct molecular patterns, where cell proliferation and gene repair mechanisms appear to play the key role.