Patterns and outcomes of late onset thyroid disturbances after COVID-19 vaccination: A report of 75 cases.

Isolated cases of subacute thyroiditis exist in the early period of COVID-19 vaccination, largely after mRNA vaccines. Here we report late onset thyroid disturbances and persistent health issues in patients of thyroid disorders after COVID-19 vaccination. Seventy-five patients with post COVID-19 vaccination thyroid disturbances were identified. Among these, 41 had flare of underlying thyroid illness, majority occurring at a median time lag of 28.4 weeks since 2nd dose. Thirty-one cases of new onset hypothyroidism and three of new onset hyperthyroidism were reported, with a median time lag respectively of 17.2 and 22.6 weeks since 2nd dose. Most cases occurred after ChAdOx1-nCoV-19, which was the commonest vaccine employed in mass roll out in India. Significant improvement was observed in majority, after a median follow up of 22-26 weeks. New onset health issues persisting for ≥4 weeks were reported in 37.3% and were common in individuals with history of COVID-19 before vaccine. New onset metabolic, musculoskeletal, and reproductive disorders were the common health complaints. Active monitoring is warranted for late onset adverse events after COVID-19 vaccines of all types. Larger studies with involvement of unvaccinated individuals are required to understand the incidence and causality of late onset thyroid disturbances after COVID-19 vaccines.

Histological Spectrum of Clinical Kidney Disease in Type 2 Diabetes Mellitus Patients with special Reference to nonalbuminuric Diabetic Nephropathy: A Kidney Biopsy-based Study.

BACKGROUND: Diabetic nephropathy (DN) is an important and catastrophic complication of diabetes mellitus (DM). Kidney disease has heterogeneity in histology in diabetes patients and includes both diabetic kidney disease (DKD) (albuminuric or nonalbuminuric) and nondiabetic kidney disease (NDKD) either in isolation or in coexistence with DN. Diabetic nephropathy is hard to overturn. While NDKD is treatable and reversible. MATERIALS AND METHODS: We enrolled a total of 50 type 2 diabetes mellitus (T2DM) patients with clinical kidney disease, of both genders and age >18 years, who underwent kidney biopsy from October 2016 to October 2018. Patients with proteinuria <30 mg per day were excluded from the study. The indications of the renal biopsy were nephrotic syndrome (NS), active urinary sediment, rapid decline in renal function, asymptomatic proteinuria, and hematuria. RESULT: A total of 50 (males: 42 and females: eight) patients with T2DM who underwent kidney biopsy were enrolled. The clinical presentation was: NS 26 (52%), chronic kidney disease (CKD) 11 (22%), asymptomatic proteinuria and hematuria six (12%), acute kidney injury (AKI) four (8%), and acute nephritic syndrome (ANS) three (6%). Diabetic retinopathy (DR) was noted in 19 (38%) cases. Kidney biopsy revealed isolated DN, isolated NDKD, and NDKD superimposed on DN in 26 (52%), 14 (28%), and 10 (20%) cases, respectively. Idiopathic membranous nephropathy (MN) (4) and amyloidosis (2) were the most common forms of NDKD, whereas diffuse proliferative glomerulonephritis (DPGN) was the main form of NDKD superimposed on DN. Diabetic nephropathy was observed in 15 (79%) cases in presence of DR and also in 11 (35.5%) cases even in absence of DR. Of eight patients with microalbuminuria four (50%) cases have biopsy-proven DN. CONCLUSION: About 48% of patients had NDKD either in isolation or in coexistence with DN. Diabetic nephropathy was found in absence of DR and in patients with a low level of proteinuria. The level of proteinuria and presence of DR does not help to distinguish DN vs NDKD. Hence, renal biopsy may be useful in selected T2DM patients with clinical kidney disease to diagnose NDKD.

Predictors of the diets consumed by adolescent girls, pregnant women and mothers with children under age two years in rural eastern India.

Adolescents, pregnant women and mothers of children under 2 years of age are in stages of life characterized by higher nutritional demands. The study measured the dietary diversity of 17,680 adolescent girls, pregnant women and mothers of children under age 2 years in the eastern Indian states of Bihar, Chhattisgarh and Odisha using data from the Swabhimaan baseline survey conducted in 2016. The association of women's mean Dietary Diversity Scores with socioeconomic, health and nutrition service indicators was assessed. The sampled population was socioeconomically more vulnerable than the average Indian population. There was not much variation in the types of foods consumed daily across target groups, with diet being predominantly cereal (98%) and vegetable (83%) based. Nearly 30% of the mothers had low Dietary Diversity Scores, compared with 25% of pregnant women and 24% of adolescent girls. In each target group, more than half of the respondents were unable to meet the Minimum Dietary Diversity score of at least five of ten food groups consumed daily. Irrespective of their background characteristics, mean Dietary Diversity Scores were significantly lower in Bihar than in Chhattisgarh and Odisha for all target groups. Having at least 6 years of education, belonging to a relatively rich household and possessing a ration card predicted mean dietary diversity. Project interventions of participatory women's group meetings improved mean Dietary Diversity Scores for mothers and adolescent girls. Considering the association between poverty and dietary diversity, the linkage between girls and women and nutrition-focused livelihoods and supplementary nutrition programmes needs to be tested.

Radiological Evaluation of Postsurgical Course of Perforators in Retrograde Posterior Tibial Fasciocutaneous Flaps for Distal Leg and Foot Defects.

Background Mapping of vascular perforators by various methodologies have been described for planning of a variety of flaps in the lower limbs. We attempted to assess the changes in posterior tibial perforators after transfer of fasciocutaneous flaps for leg defects. Methods 20 patients with distal leg and foot defects were studied by computed tomography angiography (CTA) and preoperative audio Doppler to ascertain perforators of posterior tibial artery. Fasciocutaneous flaps were raised, based on these perforators, depending on the site and size of soft-tissue defects. The number of perforators and their distance from the medial malleolus were also studied. Postoperative CTA was performed on the 7th to 10th day, with emphasis on postoperative changes of the perforators on which the flaps were based. Results One to four posterior tibial perforators were found between 5 cm and 8 cm proximal to the medial malleolus. After flap transfer, the perforators could be traced to variable distance through the total length of the flap. The perforators formed small vascular loop in 12 patients, following retrograde posterior tibial flap transfer. The height of the loop, the number of such loops, the dilatation and tortuosity of the perforators, and their longitudinal orientation were studied in detail. Most of the findings can be explained by mechanical realignment of perforators as well as by the delay phenomenon associated with retrograde fasciocutaneous flaps. Conclusion It was concluded that the morphological changes associated with the perforators explained the vascular rationality and success of these flaps.

Genome-wide differential methylation analyses identifies methylation signatures of male infertility.

STUDY QUESTION: Do methylation changes in sperm DNA correlate with infertility? STUDY ANSWER: Loss of spermatogenesis and fertility was correlated with 1680 differentially-methylated CpGs (DMCs) across 1052 genes. WHAT IS KNOWN ALREADY: Methylation changes in a number of genes have been correlated with reduced sperm count and motility. STUDY DESIGN, SIZE, DURATION: This case-control study used spermatozoal DNA from 38 oligo-/oligoastheno-zoospermic infertile patients and 26 normozoospermic fertile men. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Genome-wide methylation analysis was undertaken using 450 K BeadChip on spermatozoal DNA from six infertile and six fertile men to identify DMCs. This was followed by deep sequencing of spermatozoal DNA from 32 infertile patients and 20 fertile controls. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 1680 DMCs were identified, out of which 1436 were hypermethylated and 244 were hypomethylated. Classification of DMCs according to the genes identified BCAN, CTNNA3, DLGAP2, GATA3, MAGI2 and TP73 among imprinted genes, SPATA5, SPATA7, SPATA16 and SPATA22 among spermatogenesis-associated genes, KDM4C and JMJD1C, EZH2 and HDAC4 among genes which regulate methylation and gene expression, HLA-C, HLA-DRB6 and HLA-DQA1 among complementation and immune response genes, and CRISPLD1, LPHN3 and CPEB2 among other genes. Genes showing significant differential methylation in deep sequencing, i.e. HOXB1, GATA3, EBF3, BCAN and TCERG1L, are strong candidates for further investigations. The role of chance was ruled out by deep sequencing of select genes. LARGE-SCALE DATA: N/A. LIMITATIONS, REASON FOR CAUTION: Genome-wide analyses are fairly accurate, but may not be exactly validated in replication studies across all DMCs. We used the 't' test in the genome-wide methylation analysis, whereas other tests could provide a more robust and powerful analysis. WIDER IMPLICATIONS OF THE FINDINGS: DMCs can serve as markers for inclusion in infertility screening panels, particularly those in the genes showing differential methylation consistent with previous studies. The genes validated by deep sequencing are strong candidates for investigations of their roles in spermatogenesis. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Council of Scientific and Industrial Research (CSIR), Govt. of India with grant number BSC0101 awarded to Rajender Singh. None of the authors has any competing interest to declare.

Culture temperature modulates half antibody and aggregate formation in a Chinese hamster ovary cell line expressing a bispecific antibody.

Therapeutic bispecific antibodies are formed by assembly of multichain polypeptides. In general, a bispecific antibody has two different light chains and two different heavy chains that fold and correctly pair via engineered interchain interactions. Because of some incorrect assembly, product-related impurities can be prevalent (e.g., half molecules, mispaired light chains, homodimers), requiring its removal during subsequent purification. In this study, we investigated the modulation of impurity levels in a stable Chinese hamster ovary cell line X expressing a bispecific antibody A formed by two light chains (LC1 and LC2) and two heavy chains (HC1 and HC2) that assembled intracellularly into a heterodimer (LC1-HC1 + LC2-HC2) via engineered charged residues. Cell line X exhibited the best volumetric productivity, growth, and viability in culture compared with other clones but also showed higher levels of half antibody species (>10%); therefore, to minimize process yield loss, better understanding, and control of impurity formation was pursued. We found this cell line decreased half antibody levels from 16% to 1% when temperature changed from 36°C to 32.5°C or 31.5°C. However, lower temperature also increased high-molecular-weight (HMW) species from 4% to 12%. To determine the impurity species composition, we characterized enriched fractions with half antibody or HMW. Intact mass spectrometry analysis revealed half antibody was LC2-HC2, whereas HMW was a mixture with ~50% as LC1-HC1 homodimer. Results suggested LC2-HC2 was easily folded and could be secreted as half antibody, especially at 36°C. On the contrary, LC1-HC1 was more susceptible to misfold or aggregate, a phenomenon more acute for cell line X at lower culture temperature because of 60% increased LC1 and HC1 messenger RNA levels. Although temperature modulation was cell line X-specific, the propensity of LC2-HC2 to form half antibodies and LC1-HC1 to aggregate appeared in other cell lines also expressing bispecific antibody A, suggesting an amino-acid sequence-dependent mechanism. In summary, impurity formation in cell line X was temperature-dependent and was influenced by different molecule characteristics between the LC1-HC1 and LC2-HC2 parts. Ultimately, we selected a biphasic cell culture process with a growth phase followed by a lower temperature phase to improve product quality and purification yield.

Comparison of longitudinal changes in clinical periodontal parameters of canines and first molars treated with fixed orthodontic appliances.

INTRODUCTION: Our objective was to learn the possible effects of fixed orthodontic therapy on the periodontium of the extraction sites adjacent to canines and first molars. This was a prospective cohort study. METHODS: Sixty-two systemically healthy subjects were selected. The study was divided into the period of oral hygiene instructions and the period of active orthodontic treatment. Plaque index, gingival index, probing pocket depth, and clinical attachment loss were recorded for the canines and first molars at baseline, after 1 month of orthodontic therapy, and every 3 months until the completion of the orthodontic treatment. RESULTS: There was a statistically significant increase in all clinical parameters (plaque index, gingival index, probing pocket depth, and attachment loss; P <0.05) on the first molars compared with the canines. Significant increases in attachment loss were noted in both the canines (baseline, 0.06 ± 0.01 mm; end of treatment, 0.17 ± 0.02 mm) and the molars (baseline, 0.07 ± 0.01 mm; end of treatment, 0.20 ± 0.02 mm). CONCLUSIONS: Attachment loss cannot be explained solely by the effect of plaque or the banding of teeth. Other factors such as tooth extractions adjacent to the canines, tooth movement, and occlusal trauma may have contributing roles in the loss of periodontal support in adolescent patients.

Increased expression of endosomal members of toll-like receptor family abrogates wound healing in patients with type 2 diabetes mellitus.

The inflammatory phase of wound healing cascade is an important determinant of the fate of the wound. Acute inflammation is necessary to initiate proper wound healing, while chronic inflammation abrogates wound healing. Different endosomal members of toll-like receptor (TLR) family initiate inflammatory signalling via a range of different inflammatory mediators such as interferons, internal tissue damaged-associated molecular patterns (DAMPs) and hyperactive effector T cells. Sustained signalling of TLR9 and TLR7 contributes to chronic inflammation by activating the plasmacytoid dendritic cells. Diabetic wounds are also characterised by sustained inflammatory phase. The objective of this study was to analyse the differential expression of endosomal TLRs in human diabetic wounds compared with control wounds. We analysed the differential expression of TLR7 and TLR9 both at transcriptional and translational levels in wounds of 84 patients with type 2 diabetes mellitus (T2DM) and 6 control subjects without diabetes using quantitative real-time polymerase chain reaction (RT-PCR), western blot and immunohistochemistry. TLR7 and TLR9 were significantly up-regulated in wounds of the patients with T2DM compared with the controls and were dependent on the infection status of the diabetic wounds, and wounds with microbial infection exhibited lower expression levels of endosomal TLRs. Altered endosomal TLR expression in T2DM subjects might be associated with wound healing impairment.

A phase 2 study of bortezomib in combination with ifosfamide/vinorelbine in paediatric patients and young adults with refractory/recurrent Hodgkin lymphoma: a Children's Oncology Group study.

A Children's Oncology Group clinical trial aimed to determine if bortezomib (B) increased the efficacy of ifosfamide and vinorelbine (IV) in paediatric Hodgkin lymphoma (HL). This study enrolled 26 relapsed HL patients (<30 years) treated with two to four cycles of IVB. The primary endpoint was anatomic complete response (CR) after two cycles. Secondary endpoints included overall response (OR: CR + partial response) at study completion compared to historical controls [72%; 95% confidence interval (CI): 59-83%]. Although few patients achieved the primary objective, OR with IVB improved to 83% (95% CI: 61-95%; p = 0.32). Although not statistically different, results suggest IVB may be a promising combination.

Toll-like receptor 4 polymorphisms and their haplotypes modulate the risk of developing diabetic retinopathy in type 2 diabetes patients.

PURPOSE: Persistent inflammation and impaired neovascularization in type 2 diabetes mellitus (T2DM) patients may lead to development of macro- and microvascular complications. Diabetic retinopathy (DR) is one of the secondary microvascular complications of T2DM. Improper activation of the innate immune system may be an important contributor in the pathophysiology of DR. Toll-like receptor 4 (TLR4) is an important mediator of innate immunity, and genetic alterations in TLR4 support inflammation in the hyperglycemic condition. The present work was designed to investigate whether the TLR4 single nucleotide polymorphisms (SNPs) rs4986790, rs4986791, rs10759931, rs1927911, and rs1927914 are associated with DR in a north Indian population. METHODS: The study group of 698 individuals (128 DR, 250 T2DM, 320 controls) was genotyped by PCR-RFLP. Haplotype and linkage disequilibrium between SNPs were determined using Haploview software. RESULTS: Combined risk genotypes of TLR4 SNPs rs10759931 (odds ratio [OR] 1.50, p = 0.05) and rs1927914 (OR 1.48, p = 0.05) were found to be significantly associated with pathogenesis of DR. A total of 14 haplotypes with frequency >1% were obtained using Haploview software. Haplotypes ACATC (37.5%) and ACATT (14.8%) were the two most common haplotypes obtained. CONCLUSIONS: Results of the present case-control study that included 698 north Indian subjects suggested that TLR4 SNPs rs10759931 and rs1927914 modulate the risk of DR in T2DM cases. Association analysis using haplotypes showed none of the haplotypes were associated with either susceptibility or resistance to DR in a north Indian population.

Study of Correlation between Serum Vitamin B12 Level and Aberrant DNA Methylation in Infertile Males.

Introduction: Altered DNA methylation pattern in sperms has been associated with infertility in males demonstrating defective spermatogenesis or low semen quality. Vitamin B-12, by affecting 1-carbon metabolism pathways, might alter the DNA methylation pattern. We aimed to study the correlation of serum vitamin B12 levels with aberrant DNA methylation in infertile male patients. Methods: A cross-sectional study was conducted on 17 oligozoospermic infertile males (WHO criteria, 2010) and 10 healthy fertile males. Serum vitamin B12 levels were estimated using the chemiluminescence method. Global methylation was determined using the ELISA system (Imprint Methylated DNA Quantification Kit, Sigma-Aldrich). The levels of global DNA methylation were calculated and compared relative to the methylated (100%) control DNA provided with the kit. Results: Mean serum vitamin B12 concentration in the control group was higher than that of the case group. This difference in serum vitamin B12 concentration in both groups was found statistically significant. Although the results of this study show that oligozoospermic men have relatively lower global DNA methylation as compared to normozoospermic control, the values could not reach a statistically significant level. A small positive correlation was found between serum vitamin B12 levels and percent methylation defect (r = 0.14) but was statistically insignificant. Conclusion: Our study concludes that oligozoospermic infertile males have a significant deficiency of vitamin B12 as compared to normozoospermic fertile males. This study did not find any significant difference in global DNA methylation between the two groups. The present study does not suggest any correlation between serum vitamin B12 level and percent DNA methylation.

Congenital adrenal hyperplasia complicated by gonadotropin-dependent precocious puberty.

Precocious puberty, characterised by the early appearance of secondary sexual characteristics, poses challenges in diagnosis and management. Here, we describe a case of precocious puberty diagnosed in a boy in middle childhood, who presented with progressive phallus enlargement, pubic hair development and increased aggressive behaviour. Hormonal evaluation confirmed the diagnosis of congenital adrenal hyperplasia (CAH), complicated by gonadotropin-dependent precocious puberty. The case highlights the importance of assessment of testicular volume in a patient presenting with precocious puberty. Symmetrical testicular enlargement in a patient with CAH suggests premature activation of the hypothalamic-pituitary-gonadal axis. The patient received glucocorticoid therapy to suppress androgen production related to CAH and gonadotropin-releasing hormone analogue therapy to control premature activation of the hypothalamic-pituitary-gonadal axis. Follow-up visits showed regression of secondary sexual characteristics and improved growth velocity.

Integrated use of contrast-enhanced and grey-scale ultrasound in assigning American College of Radiology Thyroid Imaging-Reporting and Data System scores for characterisation of thyroid nodules: A prospective observational study.

Background: The advent and increased use of high-resolution ultrasonography has resulted in improved detection of thyroid nodules. Even with the use of various Thyroid Imaging-Reporting and Data System, accurate imaging diagnosis of malignant thyroid nodules has been suboptimal, which necessitated use of newer modalities like contrast-enhanced ultrasonography alone and in combination for this purpose. Although the combined use of various Thyroid Imaging-Reporting and Data System and contrast-enhanced ultrasonography has turned out to be accurate in many studies, the ideal way to integrate contrast-enhanced ultrasonography into the Thyroid Imaging-Reporting and Data System algorithm is under-investigated. Purpose: To estimate and compare the diagnostic accuracy of American College of Radiology Thyroid Imaging-Reporting and Data System and contrast-enhanced ultrasonography in differentiating benign and malignant nodules alone and in combination. To estimate the diagnostic accuracy of contrast-enhanced ultrasonography in re-categorisation of Thyroid Imaging-Reporting and Data System 3 and Thyroid Imaging-Reporting and Data System 4 thyroid nodules. Materials and methods: This was a prospective cohort study performed in a tertiary care university-based hospital for 3 years. Adult patients with clinical or previous sonographic diagnosis of thyroid nodules were selected. Each of the nodules were assessed using ultrasonography and categorised using American College of Radiology Thyroid Imaging-Reporting and Data System criteria. The lesion was then assessed for contrast-enhanced ultrasonography features. The final diagnosis of the nodules was made using fine needle aspiration cytology. The diagnostic accuracy in diagnosis of malignant thyroid nodules for each of the American College of Radiology Thyroid Imaging-Reporting and Data System and contrast-enhanced ultrasonography alone and in combination was assessed. The diagnostic accuracy of contrast-enhanced ultrasonography in diagnosis of malignant thyroid nodules categorised as Thyroid Imaging-Reporting and Data System 3 and Thyroid Imaging-Reporting and Data System 4 was also assessed. Results: American College of Radiology Thyroid Imaging-Reporting and Data System had a sensitivity, specificity, negative predictive value, positive predictive value and diagnostic accuracy of 86.6%, 54.5%, 17.4%, 97.3% and 57.7%, respectively, in diagnosis of malignant thyroid nodules. Contrast-enhanced ultrasonography had a sensitivity, specificity, negative predictive value, positive predictive value and diagnostic accuracy of 86.6%, 95.4%, 67.9%, 98.4% and 94.4%, respectively, in diagnosis of malignant thyroid nodules. Contrast-enhanced ultrasonography had sensitivity, specificity, negative predictive value, positive predictive value and diagnostic accuracy of 93.3%, 100.0%, 100.0%, 99.2% and 99.3%, respectively, in re-categorisation of Thyroid Imaging-Reporting and Data System 3 and Thyroid Imaging-Reporting and Data System 4 nodules. Conclusion: Contrast-enhanced ultrasonography can play a key role in diagnosis of malignant thyroid nodules which are categorised as indeterminate on grey-scale ultrasound.

Quality of Life Assessment and Comparative Analysis of Anterolateral Thigh Flap and Pectoralis Major Myo-cutaneous Flap for Reconstruction of Post Excision Defects of Oral Squamous Cell Carcinoma.

Present study is a comparative evaluation of the financial impact on choice of flap, clinical results as well as post resection quality of life assessment of cancer patients reconstructed with anterolateral thigh(ALT) flap and pectoralis major myo-cutaneous(PMMC) flap. Ethical clearance was obtained from institutional ethical committee. In this non-randomised, prospective study design among 49 oral squamous cell carcinoma cases, 39 cases were reconstructed using PMMC flap and 10 using ALT flap from year 2020 to 2022. These 2 flaps were compared in terms of parameters like time utilised for reconstruction, hospital stay, overall survival and Washington University Quality of Life index(UW-QOL) for head and neck cancer. Patients were followed regularly for post-operative complications. The University of Washington Quality of Life score (UW-QOL) questionnaire, version 4, was completed at six months post-operatively. Obtained data was statistically analysed using IBM® SPSSⓇ. Washington University Quality of Life index scores and esthetics are better with Antero-lateral thigh flap, with less overall post-operative complications. On the other hand Pectoralis myocutaneous flap has less surgical time and more salvage potential. Despite of Antero-lateral thigh flap having Good Washington University Quality of Life index scores, lesser post-operative complications and better esthetics; scale of developing nations is still tipping towards economical, less time consuming and less technique sensitive Pectoralis major myocutaneous flap.

Molecular study of the KCNJ11 gene and its correlation with Prakriti to preventing and managing type 2 diabetes.

In Ayurveda, every individual is believed to possess a unique entity known as Prakriti, which distinguishes them from others physically, physiologically, and psychologically. This entity also determines an individual's response to a particular stimulus, and it is believed that such responses are not solely determined by genetics. The present research aims to validate the Ayurvedic concept of Prakriti from a modern molecular perspective to strengthen the personalized and precise treatment approach. A study was conducted to investigate the role of the KCNJ11gene in the susceptibility of individuals to type 2 diabetes mellitus (T2DM) with their metabolic status. The research involved allele mining on three major Prakriti groups - Vata, Pitta, and Kapha - in 112 patients with T2DM and 112 healthy individuals. The KCNJ11 gene, responsible for insulin secretion membrane pore formation, was analyzed to determine the susceptibility of different Prakriti types to T2DM. The MutPred tool predicted the molecular cause of disease-related amino acid substitution. According to the study, only Pitta and Kapha Prakriti were diagnosed with diabetes, while all three Prakriti types were present in the control group of healthy individuals. A protein model was prepared, and the changes resulting from mutations were observed for each group in their protein sequence, both as synonymous and non-synonymous mutations. Ultimately, these changes contributed to the manifestation of T2DM. Based on the findings, it appears that Prakriti groups may experience changes in protein function due to nonsynonymous mutations and differences in amino acids at the protein level.

Fast and scalable purification of a therapeutic full-length antibody based on process crystallization.

The potential of process crystallization for purification of a therapeutic monoclonal IgG1 antibody was studied. The purified antibody was crystallized in non-agitated micro-batch experiments for the first time. A direct crystallization from clarified CHO cell culture harvest was inhibited by high salt concentrations. The salt concentration of the harvest was reduced by a simple pretreatment step. The crystallization process from pretreated harvest was successfully transferred to stirred tanks and scaled-up from the mL-scale to the 1 L-scale for the first time. The crystallization yield after 24 h was 88-90%. A high purity of 98.5% was reached after a single recrystallization step. A 17-fold host cell protein reduction was achieved and DNA content was reduced below the detection limit. High biological activity of the therapeutic antibody was maintained during the crystallization, dissolving, and recrystallization steps. Crystallization was also performed with impure solutions from intermediate steps of a standard monoclonal antibody purification process. It was shown that process crystallization has a strong potential to replace Protein A chromatography. Fast dissolution of the crystals was possible. Furthermore, it was shown that crystallization can be used as a concentrating step and can replace several ultra-/diafiltration steps. Molecular modeling suggested that a negative electrostatic region with interspersed exposed hydrophobic residues on the Fv domain of this antibody is responsible for the high crystallization propensity. As a result, process crystallization, following the identification of highly crystallizable antibodies using molecular modeling tools, can be recognized as an efficient, scalable, fast, and inexpensive alternative to key steps of a standard purification process for therapeutic antibodies.

IgG1 and IgG4 antibodies sample initial structure dependent local conformational states and exhibit non-identical Fab dynamics.

We have investigated the dynamics of two [Formula: see text]-immunoglobulin molecules, IgG1 and IgG4, using long all atom molecular dynamics simulations. We first show that the de novo structures of IgG1 and IgG4 predicted using AlphaFold, with no interactions between the fragment crystallizable (Fc) domain and the antigen fragment binding domain (Fab), eventually relaxes to a state with persistent Fc-Fab interactions that mirrors experimentally resolved structures. We quantified the conformational space sampled by antibody trajectories spawned from six different initial structures and show that the individual trajectories only sample states bound by a local minimum and display very little mixing in their conformational states. Furthermore, the dynamics of the individual Fab domains are strongly dependent on the initial crystal structure and isotype. In all conditions, we observe non-identical dynamics between the Fab arms in an antibody. For a six-bead coarse grained model, we show that non-covalent Fc-Fab interactions can modulate the stiffnesses associated with Fc-Fab distances, angles, and dihedral angles by up to three orders of magnitude. Our results clearly illustrate the inherent complexities in studying antibody dynamics and highlight the need to include non-identical Fab dynamics as an inherent feature in computational models of therapeutic antibodies.

Heterogeneity in Disulfide Bond Reduction in IgG1 Antibodies Is Governed by Solvent Accessibility of the Cysteines.

We studied unpaired cysteine levels and disulfide bond susceptibility in four different γ-immunoglobulin antibodies using liquid chromatography-mass spectrometry. Our choice of differential alkylating agents ensures that the differential peaks are non-overlapping, thus allowing us to accurately quantify free cysteine levels. For each cysteine residue, we observed no more than 5% to be unpaired, and the free cysteine levels across antibodies were slightly higher in those containing lambda light chains. Interchain and hinge residues were highly susceptible to reducing stresses and showed a 100-1000-fold higher rate of reduction compared to intrachain cysteines. Estimations of the solvent-accessible surface for individual cysteines in IgG1, using an implicit all-atom molecular dynamics simulation, show that interchain and hinge cysteines have >1000-fold higher solvent accessibility compared to intrachain cysteines. Further analyses show that solvent accessibility and the rate of reduction are linearly correlated. Our work clearly establishes the fact that a cysteine's accessibility to the surrounding solvent is one of the primary determinants of its disulfide bond stability.

The Prevalence of Anemia in Hospitalized Patients With Diabetic Foot Ulcer (DFU) and the Relationship Between the Severity of Anemia and the Severity of DFU.

BACKGROUND AND AIMS: We aim to determine the prevalence of anemia in hospitalized patients with diabetic foot ulcers (DFUs) and estimate the relationship between the severity of anemia and diabetic foot ulcer. MATERIALS AND METHODS: We retrospectively collected and evaluated the data of 323 patients hospitalized with diabetic foot ulcer (DFU). We included 299 type 2 diabetic patients with foot ulcers of neuropathic or neuroischemic nature with infection. Anemia was defined based on World Health Organization (WHO) criteria, and the severity of DFU was classified in University of Texas (UT) grades. RESULTS: Anemia was detected in 94.3% of DFU, and the prevalence of mild, moderate, and severe anemia was 16.7%, 55.7%, and 27.6%, respectively. There was a significant difference in the mean hemoglobin (Hb) levels among the patients with varying grades of severity of DFU (1B: Hb=10.17±2.08 gm/dL, 2B: Hb=9.27±2.04 gm/dL, 3B: Hb=8.03±1.829 gm/dL; p value=<0.0001). The iron study was available in 141 (47.15%) patients and was suggestive of anemia of chronic disorder (mean serum iron=40.22±23.81 mcg/dL, mean total iron-binding capacity (TIBC)=239.34±67.24 mcg/dL, mean ferritin=378.05±141.337 ng/mL). TIBC significantly decreased (1B=262.13±61.05, 2B=233.65±71.26, 3B=222.43±74.18; p=0.04), and ferritin significantly increased (1B=309.9±70.76, 2B=351.73±94.22, 3B=488.58±170.4; p<0.0001) with increasing DFU severity. Hemoglobin was significantly decreased at the time of discharge in comparison to that at admission (9.3±2.1 gm/dL versus 8.8±1.5 gm/dL; p value=0.01). Red blood cell (RBC) counts, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), lymphocyte counts, albumin, calcium, and high-density lipoprotein (HDL) significantly decreased with the increase of DFU severity. The duration of hospitalization, total leucocyte counts, neutrophil counts, and neutrophil-to-lymphocyte ratio (NLR) increased with the severity of DFU. CONCLUSIONS: The prevalence of anemia was very high in DFU and more than three-fourths of the patients had moderate to severe anemia. The severity of anemia was associated with the severity of DFU. The most common cause of anemia was anemia of chronic disorder secondary to diabetic foot infection. During the period of hospitalization, hemoglobin decreased despite improvement in DFU infection.

Quantifying the margin sharpness of lesions on radiological images for content-based image retrieval.

PURPOSE: To develop a method to quantify the margin sharpness of lesions on CT and to evaluate it in simulations and CT scans of liver and lung lesions. METHODS: The authors computed two attributes of margin sharpness: the intensity difference between a lesion and its surroundings, and the sharpness of the intensity transition across the lesion boundary. These two attributes were extracted from sigmoid curves fitted along lines automatically drawn orthogonal to the lesion margin. The authors then represented the margin characteristics for each lesion by a feature vector containing histograms of these parameters. The authors created 100 simulated CT scans of lesions over a range of intensity difference and margin sharpness, and used the concordance correlation between the known parameter and the corresponding computed feature as a measure of performance. The authors also evaluated their method in 79 liver lesions (44 patients: 23 M, 21 F, mean age 61) and 58 lung nodules (57 patients: 24 M, 33 F, mean age 66). The methodology presented takes into consideration the boundary of the liver and lung during feature extraction in clinical images to ensure that the margin feature do not get contaminated by anatomy other than the normal organ surrounding the lesions. For evaluation in these clinical images, the authors created subjective independent reference standards for pairwise margin sharpness similarity in the liver and lung cohorts, and compared rank orderings of similarity used using our sharpness feature to that expected from the reference standards using mean normalized discounted cumulative gain (NDCG) over all query images. In addition, the authors compared their proposed feature with two existing techniques for lesion margin characterization using the simulated and clinical datasets. The authors also evaluated the robustness of their features against variations in delineation of the lesion margin by simulating five types of deformations of the lesion margin. Equivalence across deformations was assessed using Schuirmann's paired two one-sided tests. RESULTS: In simulated images, the concordance correlation between measured gradient and actual gradient was 0.994. The mean (s.d.) and standard deviation NDCG score for the retrieval of K images, K = 5, 10, and 15, were 84% (8%), 85% (7%), and 85% (7%) for CT images containing liver lesions, and 82% (7%), 84% (6%), and 85% (4%) for CT images containing lung nodules, respectively. The authors' proposed method outperformed the two existing margin characterization methods in average NDCG scores over all K, by 1.5% and 3% in datasets containing liver lesion, and 4.5% and 5% in datasets containing lung nodules. Equivalence testing showed that the authors' feature is more robust across all margin deformations (p < 0.05) than the two existing methods for margin sharpness characterization in both simulated and clinical datasets. CONCLUSIONS: The authors have described a new image feature to quantify the margin sharpness of lesions. It has strong correlation with known margin sharpness in simulated images and in clinical CT images containing liver lesions and lung nodules. This image feature has excellent performance for retrieving images with similar margin characteristics, suggesting potential utility, in conjunction with other lesion features, for content-based image retrieval applications.