Evaluation of salt content and effectiveness of excessive salt reduction methods in selected commercially available dried fish types in Sri Lanka

High salt intake elevates the risk of non-communicable diseases such as high blood pressure, cardiovascular diseases and stroke worldwide. Sri Lanka has recorded in 2010 as the country with highest average fish & fish products consumption in South Asia. In the current study, salt in ten types of commonly available dried fish namely; sprats, prawns, smoothbelly sardinella, queen fish, cat fish, sail fish, shark, skipjack tuna, Bombay duck and trenched sardinella was analyzed and determined salt reduction methods with minimal protein loss. Four salt reduction methods were tested; Method 1: washed with water at Room Temperature (RT) for five times; Method 2: washed with water for five times at RT and kept in hot water for 5min; Method 3: washed with water for five times at RT and boiled for 5min; Method 4: washed with water for five times at RT and kept in coconut water for 5min. Using Volhard method, sodium chloride was analyzed while protein was determined using Kjeldahl method. All four methods showed significant reduction of salt level in tested dried fish (p < 0.05). Among the tested salt reduction methods, Method 3 showed the highest salt reduction for all types of dried fish except smoothbelly sardinella and cat fish.The highest salt mean value of 28.8% was recorded in queen fish and the lowest mean value of 12.8% was recorded in smoothbelly sardinella. The highest protein loss was recorded in Method 3. To reduce considerable amount of salt, the easiest and fairly effective method is method 1 except for prawns and Shark. Although higher salt reduction showed in method 2 and 3, they are not recommended due to high protein loss, high energy expenditure and reduction of freshness of dried fish. Method 4 can be applied for all dried fish types because it is economical and reduces considerable amount of salt, removes less amount of protein comparatively. The results revealed that all tested dried fish except smoothbelly sardinella contain significantly high amount of salt (p < 0.05) than the standard value specified (12%) by the Sri Lanka Standards Institution (SLSI).Keywords: Dried fish, protein loss, salt-intake, salt reduction, non-communicable diseases

Study of Prevalance of Nephropathy among Sickle Cell Disease Patients in Waghodia Region, Vadodara, Gujarat

Introduction: Sickle-cell disease (or drepanocytosis) is a life-long blood disorder Characterized by red blood cells that assume an abnormal, rigid, sickle shape. Sickle cell disease (SCD) has several complications, including chronic renal failure, manifesting with hypertension (high blood pressure) proteinuria (protein loss in the urine), hematuria (redblood cells in urine) and worsening anaemia. Progression to end-stage renal failure confers a poor prognosis. Objective: The objective of the study was to determine the Prevalence of Nephropathy among sickle cell disease patients. Materials and Methods: This cross sectional study includes a total 150 participants who suffering from sickle cell anemia and attending our Institute. Renal function test and Urine examination of all participants was done. Estimated Glomerular Filtration Rate (eGFR) calculated using the Cockroft Gault formula. Comparison of results was done between Sickle cell trait and Sickle cell disease Group. Results: The mean age of the SCA patients were 25.54±10 years. Maximum participants are found to be from age group 25-30 yr(n=35) followed by 20-25 yr(n=30). Of the 150 SCA patients, 89 (59.33%), and 61 (40.66%) were males and females, respectively. The Mean value of S.Creatinine of SCT group is 0.73±0.46 mg/dl and SCD is 1.0±0.35 mg/dl, while the Mean value of eGFR is 134.19±87.21 ml/min and 124.20 ±58.25 ml/min in SCT and SCD Group respectively. Conclusions: From our study we conclude that the Derangement of Kidney function in sickle cell disease is frequent in our setting especially among young adult. It concerns SCD as well as SCT patients. Albuminuria is more frequent in homozygote patients and its prevalence increase with age. Age ≥ 25 years is associated with high risk of CKD in SCA group and albuminuria in SCD.

Diagnostic utility of protein excretion in 24-hour urine and the protein to creatinine ratio for adverse perinatal outcomes and time of delivery

Objective Our study aimed to evaluate the perinatal and neonatal outcomes of hypertensive pregnant women with or without proteinuria. We compared the predictivity of spot urinary protein to creatinine (P/C) ratio and 24-hour protein excretions on outcomes. Methods We retrospectively enrolled 230 pregnant women with a new diagnosis of hypertension between 20 and 37 weeks of gestation. We divided the patients into two groups according to the protein level determined by 24-hour urine collection and P/C ratio. The presence and level of proteinuria, its relationship with the P/C ratio, and the relationship between these findings and perinatal outcomes were evaluated. Results Gestational age at delivery weeks and latency period (duration between diagnosis of hypertension and delivery) were significantly earlier in pregnant women with ≥300 mg/24-h and P/C ratio ≥0.3. Adverse neonatal outcomes were significant in patients with proteinuria ≥300 mg/24-hour and P/C ratio ≥0.3. Urinary protein levels in 24-hour urine were significantly higher in pregnant women with P/C ratio ≥0.3 and a significantly positive correlation was found between 24-h proteinuria and P/C (r=0.382, p<0.001). Conclusion Our study demonstrated that a protein loss of ≥300 mg in 24-h and a P/C ratio in spot urine ≥0.3 in hypertensive pregnant women is associated with adverse perinatal outcomes. Furthermore, we have identified that proteinuria ≥300 mg/day and spot urine P/C ratio ≥0.3 in hypertensive pregnant women make them prone to early delivery risk.

Epigenetic Alteration and its Association With Downregulated FOXP3 Gene in Indian Breast Cancer Patients

Background:FOXP3 gene, known to be a potential tumor suppressor, has been identified to interact with HER2 in mammary cancer. Moreover, the high expression of FOXP3 serves as a good predictor of the survival of patients in breast cancer, prostate cancer, and gastric cancer. The expression and epigenetic alterations were evaluated in female breast cancer patients.Material and Methods: Expression studies at the mRNA level and protein level were conducted in 140 breast cancer cases by real-time PCR and immunohistochemistry, respectively. Epigenetic studies were also conducted by analyzing the methylation status at the promoter region of the gene using MS-PCR.Results:FOXP3 mRNA expression and protein expression were downregulated in breast cancer patients. The absence of FOXP3 protein expression is significantly associated with promoter methylation, where 70 methylated cases exhibited protein loss (70/95, 73.6%). Statistically, we also found a significant correlation between FOXP3 protein expression and TNM stage, promoter methylation, and histological grade. The methylated FOXP3 cases that did not express protein were also significantly associated with positive lymph node metastasis and HER-2 status.Conclusion: The expression profile of FOXP3 may serve as a prognostic factor. In short, FOXP3 may stand in the most crucial list of biomarkers for breast cancer, bringing compelling results in terms of treatment and management of the disease.

Processing Human Milk to Increase Nutrient Density for Preterm Infants

Background: Human milk is the optimal food for newborns. Choices to feed preterm infants in neonatal intensive care units are mother’s milk, donor milk, or formula. Preterm infants have better tolerance for human milk, but the lower caloric density of donor milk might not meet preterm infant growth needs. Preterm infants have higher protein and energy requirements with a limited stomach capacity. Therefore, there is a need for human milk with increased nutrient density. Research Aim: To concentrate donor milk to have a higher caloric and protein density while avoiding side effects of high osmolality by precipitating lactose at low temperatures. Methods: We investigated the results of volume reduction and lactose removal processes on the lactose, protein, osmolality, and viscosity of human milk. Donor milk was obtained from WakeMed Mothers’ Milk Bank. Homogenization and evaporative condensation were applied to samples ( N = 36) before they were stored frozen overnight, followed by refrigerated centrifugation for lactose removal at 0 °C. Supernatants were separated and compared to the composition of controls. Results: A significant reduction of lactose ( SW = -262, p < .0001) and osmolality ( SW = -211.5 p < .01) was achieved in the concentrated milk without a significant protein loss from centrifugation ( SW = -44.5, p = .49). A 30%–40% volume reduction is within the American Academy of Pediatrics recommended osmolality for infant feeding. Conclusion: Concentrating human milk in a milk bank setting for feeding preterm infants might be a simple and low-cost process to achieve a product with higher nutrient density and no non-human components.

Loss of COP9 Signalosome Gene-Containing 2q Region Is Associated with Lenalidomide and Pomalidomide Resistance in Myeloma Patients

Introduction Identification of the causes of, and biomarkers for, drug resistance in myeloma is important for understanding treatment failures, and for future instigation of targeted therapeutics for myeloma. Using the largest set of whole genome sequencing (WGS) of advanced and drug resistant multiple myelomas to date, we reported that even heterozygous loss of the 3p region, which harbours immunomodulatory drug (IMiD) and CRBN E3 ligase modulator drug (CELMoD)-binding protein Cereblon (CRBN), undergoes strong therapeutic selection on lenalidomide (LEN) and/or pomalidomide (POM) treatment (Gooding et al 2021, PMC7893409). We hypothesized that copy loss of other genes required for IMiD activity may also have clinical relevance. Several groups have reported pharmacogenetic screens identifying genes essential for IMiD sensitivity in vitro, particularly genes required for the maintenance of the CUL4-DDB1-CRBN E3 Ubiquitin Ligase, such as members of the COP9 signalosome complex, function of which prevents CRBN protein degradation. However, loss of these genes has hitherto not been reported in myeloma. Methods and results We identified candidate genes whose loss may favor IMiD drug resistance from published pharmacogenetic screens (n=5), and shortlisted genes consistently identified as essential for LEN or POM function in ≥2 screens (n=23). In our WGS dataset of 455 patients (cohorts: newly diagnosed (ND) n = 198, LEN-refractory n = 203; and LEN-then-POM-refractory n = 54), the incidence of mutation of shortlisted LEN/POM-essential genes in drug-refractory cohorts was rare (&lt;5% combined), as found with CRBN. We next identified all those with overall incidence of &gt;10% copy loss at the LEN-then-POM-refractory state, plus incidence of copy loss that increased from ND to LEN-then-POM-refractory states by ≥1.5-fold. This delivered 3 copy loss regions for further investigation: a) 3p, which we had already reported; b) 17p, loss of which is known to be strongly selected in myeloma as the site of TP53; and c) 2q, previously unidentified as relevant in myeloma, but whose minimal common region contained two members of the COP9 signalosome (COPS7B, COPS8). Proportion of loss of this region increased between ND (5.5%), LEN-refractory (9.8%) and LEN-then-POM-refractory states (16.6%), p=0.009. Those patients who had lost a copy of these genes also demonstrated a significant reduction in COPS7B/COPS8 gene expression (p&lt;0.01 both genes). In a separate cohort of myeloma patients (n=24) with sequential sample WGS analysis before and after LEN and/or POM resistance acquisition, we traced acquisition of CNA-defined subclones. 5/24 (21%) patients had acquired either clonal or subclonal loss of the 2q region containing COPS7B and COPS8 at IMID resistance, which had been either absent or below limit of detection pre-IMiD exposure. No other CNA newly-emerged in such a high proportion during IMiD treatment. Relative decrease in even one COP9 signalosome gene has been shown to cause CRBN protein level to fall, and reduce LEN efficacy (Sievers et al 2018, PMC6148446). We are now analysing CRBN protein levels in sequential biopsies from these cases. Conclusion Copy number aberrations have not previously been shown to drive a therapy-specific clonal advantage in myeloma in the clinic. We have now identified a second novel CNA, 2q loss, which increases in incidence through LEN- and POM-refractory states to emerge as a marker of dominant clones in advanced, IMiD-resistant disease. Whether these CNAs will mark resistance to novel CELMoDs remains to be seen. The CRBN protein is key to the function of these drugs, and many novel proteolysis targeting chimeras (PROTACs) in development, but whether the kinetics of their CRBN binding are as sensitive to relative CRBN protein loss remains a key question. CNAs may be easily and cost-effectively detected in the clinic by targeted sequencing approaches, and may prove valuable in future therapeutic decision making. Disclosures Gooding: Bristol Myers Squibb: Research Funding. Ansari-Pour: Bristol Myers Squibb: Consultancy. Karagoz: h.: Research Funding. Ortiz Estevez: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Towfic: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Flynt: BMS: Current Employment, Current equity holder in publicly-traded company. Pierceall: BMS: Current Employment, Current equity holder in publicly-traded company. Yong: Sanofi: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; GSK: Honoraria; Amgen: Honoraria; BMS: Research Funding; Autolus: Research Funding. Vyas: Astellas: Consultancy, Honoraria; Takeda: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Gilead: Honoraria; Jazz: Honoraria; AbbVie: Consultancy, Honoraria. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

PHF6 Positively Regulates Transcription of Myeloid Differentiation Genes By Binding at Enhancer Regions

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by uncontrolled division, and differentiation arrest, of hematopoietic stem cells (HSCs) and myeloid progenitors. AML is a genetically heterogeneous disease, with mutations in genes belonging to multiple functional groups. Of these, chromatin regulators and transcriptional factors (TFs) are an important functional group to study because of a lack of targeted AML therapies against these factors. PHD Finger Protein 6 (PHF6) is one such chromatin-associated protein with a yet-unknown molecular mechanism of action. It is mutated in 3-5% of MDS, CMML, and AMLs and 20% of T- ALLs, and is considered a leukemia suppressor. To assess the effects of PHF6 on the AML transcriptome, we generated CRISPR-mediated knockout (KO) clones in THP-1 monocytic AML cell line, and integrated a Dox-inducible PHF6 construct into a PHF6 KO clone, enabling us to conditionally rescue PHF6 expression to parental levels (Fig A & B). RNA-Seq analysis of these knockout and rescue systems revealed that PHF6 expression upregulates genes related to myeloid differentiation and downregulates genes related to hematopoietic stem cells and cell division in myeloid cells. Additionally, loss of PHF6 increased THP-1 proliferation, and restoring PHF6 expression decreased proliferation. We also performed ChIP-Seq for PHF6 in the THP-1 cells and found that PHF6 binds to open and active enhancer regions. Unbiased motif analysis showed that PHF6-bound enhancers (compared to all enhancers genome-wide) were enriched for RUNX1, PU.1, and IRF8 motifs. Metagene plotting of PHF6 ChIP-Seq signal against ChIP-Seq for these TFs showed striking concordance in their binding patterns, indicating that PHF6 co-occupies chromatin with key hematopoietic transcription factors. (Fig C). PHF6 has two extended PHD (ePHD) domains with a similar structure to canonical PHD domains, but with unknown functions. Based on leukemia genome sequencing results from COSMIC, we observed that while nonsense and frameshift mutations of PHF6 (accounting for 2/3 rd of PHF6 mutations, and expected to produce no protein) are distributed throughout the gene body, missense mutations (accounting for 1/3 rd of PHF6 mutations and expected to produce a full-length protein with single amino acid substitution), are concentrated in the second ePHD domain (ePHD2). To assess the functional consequence of mutations in the ePHD2 domain, we generated from a PHF6 KO clone a clone expressing Dox-inducible PHF6 R274Q, the most common missense mutation of PHF6 seen in leukemia. RNA-Seq showed that PHF6 R274Q induction has no downstream effects on the cellular transcriptome, in striking contrast to the effects of wildtype PHF6 induction (Fig D). Additionally, the expression of PHF6 R274Q has no effect on cell growth. These results indicate that the ePHD2-domain mutant PHF6 R274Q is functionally dead (fully or partially), and the ePHD2 domain is critical for PHF6 function. Our results support an important transcriptional role of PHF6 in the myeloid system, involving co-occupancy with TFs at enhancers to promote the transcription of myeloid differentiation genes. This loss of this transcriptional regulation, either through complete PHF6 protein loss or point mutation of its ePHD2 domain, dysregulates myeloid differentiation and contributes to leukemogenesis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Regulation of Endoplasmic Reticulum–Mitochondria Tethering and Ca2+ Fluxes by TDP-43 via GSK3β

Mitochondria–ER contacts (MERCs), tightly regulated by numerous tethering proteins that act as molecular and functional connections between the two organelles, are essential to maintain a variety of cellular functions. Such contacts are often compromised in the early stages of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). TDP-43, a nuclear protein mainly involved in RNA metabolism, has been repeatedly associated with ALS pathogenesis and other neurodegenerative diseases. Although TDP-43 neuropathological mechanisms are still unclear, the accumulation of the protein in cytoplasmic inclusions may underlie a protein loss-of-function effect. Accordingly, we investigated the impact of siRNA-mediated TDP-43 silencing on MERCs and the related cellular parameters in HeLa cells using GFP-based probes for MERCs quantification and aequorin-based probes for local Ca2+ measurements, combined with targeted protein and mRNA profiling. Our results demonstrated that TDP-43 down-regulation decreases MERCs density, thereby remarkably reducing mitochondria Ca2+ uptake after ER Ca2+ release. Thorough mRNA and protein analyses did not highlight altered expression of proteins involved in MERCs assembly or Ca2+-mediated ER–mitochondria cross-talk, nor alterations of mitochondrial density and morphology were observed by confocal microscopy. Further mechanistic inspections, however, suggested that the observed cellular alterations are correlated to increased expression/activity of GSK3β, previously associated with MERCs disruption.

Dry Powders for Inhalation Containing Monoclonal Antibodies Made by Thin-Film Freeze-Drying

Thin-film freeze-drying (TFFD) is a rapid freezing and then drying technique used to prepare inhalable dry powders from the liquid form for drug delivery to the lungs. We report the preparation of aerosolizable dry powders of monoclonal antibodies (mAbs) by TFFD. We first formulated IgG with lactose/leucine (60:40 w/w) or trehalose/leucine (75:25). IgG 1% (w/w) formulated with lactose/leucine (60:40 w/w) in phosphate buffered saline (PBS) (IgG-1-LL-PBS) and processed by TFFD was found to produce the powder with the most desirable aerosol properties. We then replaced IgG with a specific antibody, anti-programmed cell death protein (anti-PD-1 mAb), to prepare a dry powder (anti-PD1-1-LL-PBS), which performed similarly to the IgG-1-LL-PBS powder. The aerosol properties of anti-PD1-1-LL-PBS were significantly better when TFFD was used to prepare the powder as compared to conventional shelf freeze-drying (shelf FD). The dry powder had a porous structure with nanoaggregates. The dry powder had a Tg value between 39-50 degree Celsius. When stored at room temperature, the anti-PD-1 mAb in the TFFD powder was more stable than that of the same formulation stored as a liquid. The addition of polyvinylpyrrolidone (PVP) K40 in the formulation was able to raise the Tg to 152 degree Celsius, which is expected to further increase the storage stability of the mAbs. The PD-1 binding activities of the anti-PD-1 mAbs before and after TFFD were not different. While protein loss, likely due to protein binding to glass or plastic vials and the TFF apparatus, was identified, we were able to minimize the loss by increasing mAb content in the powders. Lastly, we show that another mAb, anti-TNF-alpha;, can also be converted to a dry powder with a similar composition by TFFD. We conclude that TFFD can be applied to produce stable aerosolizable dry powders of mAbs for pulmonary delivery.