Middle-Down Fragmentation for the Identification and Quantitation of Site-Specific Methionine Oxidation in an IgG1 Molecule

A middle-down LC/MS approach, for the rapid quantitation and characterization of site-specific methionine oxidation in a recombinant monoclonal IgG1 molecule, is described. An IgG1 antibody was digested with endoprotease LysC under limited proteolytic conditions to produce two major components; an antigen binding fragment (Fab) and a crystallizable fraction (Fc). These fractions were then reduced to produce three major species; light chain (LC), Fc/2 which is the C terminal region of the heavy chain (HC) and the N-terminal heavy chain region (Fd). These three fragments were separated by reversed-phase HPLC using a diphenyl column. The diphenyl column resolved site-specific methionine oxidation in all three subunits. Middle- down N-terminal sequencing with a LCT premier mass spectrometer was used to identify the sites of oxidation in the LC. Sites of oxidation in the Fc/2 were identified using middle-down collision-induced dissociation (CID) on a Qtof premier. This method allowed for the rapid quantitation and identification of oxidation on each methionine residue in an IgG1 molecule.     

Mast cell tryptase may modulate endothelial cell phenotype in healing myocardial infarcts

Mast cells and macrophages infiltrate healing myocardial infarcts and may play an important role in regulating fibrous tissue deposition and extracellular matrix remodelling. This study examined the time-course of macrophage and mast cell accumulation in healing infarcts and studied the histological characteristics and protease expression profile of mast cells in a canine model of experimental infarction. Although macrophages were more numerous than mast cells in infarct granulation tissue, macrophage density decreased during maturation of the scar, whereas mast cell numbers remained persistently elevated. During the inflammatory phase of infarction, newly recruited leucocytes infiltrated the injured myocardium and appeared to be clustered in close proximity to degranulating cardiac mast cells. During the proliferative phase of healing, mast cells had decreased granular content and were localized close to infarct neovessels. In contrast, macrophages showed no selective localization. Mast cells in healing canine infarcts were alcian blue/safranin-positive cells that expressed both tryptase and chymase. In order to explain the pro-inflammatory and angiogenic actions of tryptase--the major secretory protein of mast cells--its effects on endothelial chemokine expression were examined. Chemokines are chemotactic cytokines that play an important role in leucocyte trafficking and angiogenesis and are highly induced in infarcts. Tryptase, a proteinase-activated receptor (PAR)-2 agonist, induced endothelial expression of the angiogenic chemokines CCL2/MCP-1 and CXCL8/IL-8, but not the angiostatic chemokine CXCL10/IP-10. Endothelial PAR-2 stimulation with the agonist peptide SLIGKV induced a similar chemokine expression profile. Mast cell tryptase may exert its angiogenic effects in part through selective stimulation of angiogenic chemokines.     

Utilizing the Retrograde Flow of Internal Mammary Vessels as a Donor Pedicle

Introduction  The aim of this study is to assess the results of retrograde flow of internal mammary artery and vein (IMA/V) as a donor vessel for free flap microvascular anastomosis (MVA). This need arises with bipedicle deep inferior epigastric perforator (DIEP) flaps, when all four zones with extra fat need to be harvested for unilateral breast reconstruction coupled with poor midline crossover of circulation naturally or because of midline scar. Large anterolateral thigh flaps for chest wall cover, with multiple perforators from separate pedicles, also need supercharging. This needs an additional source of donor vessels, antegrade IMA/V being the first one. Materials and Methods  Retrospective study of microvascular breast reconstruction using retrograde internal mammary donor vessels. Results  Out of 35 cases, 20 cases had distal IMA/V, with retrograde flow, as donor vessel for second set of arterial and venous anastomosis. In two cases, retrograde IMA/V was used for the solitary set of MVA. In remaining 13 cases, either retrograde IMA or V was utilized either as a principal or accessory donor. No flap was lost. Venous and arterial insufficiency happened in one case each, both were salvaged. Two cases developed partial necrosis, needing debridement and suturing. One case developed marginal necrosis. Only one case developed fat necrosis with superadded infection on follow-up. Conclusion  Distal end of IMA and IMV on retrograde flow is safe for MVA as an additional or sole pedicle. It is convenient to use being in the same field. It enables preservation of other including thoracodorsal pedicle and latissimus dorsi flap for use in case of a complication or recurrence.     

Screening for gestational diabetes,Ahmedabad, India

ObjectiveTo implement a community-based screening and awareness-raising project for gestational diabetes in Ahmedabad, India.MethodsThe project took place between April 2016 and August 2019 in Ahmedabad. Medical college faculty members and medical officers trained 3582 paramedical staff on screening for gestational diabetes. These paramedical staff tested all pregnant women 24–28 weeks gestation, who were attending village health and nutrition days – also called mamta days – in urban and rural health centres for routine antenatal care, for gestational diabetes. An oral glucose tolerance test was used and blood sugar ≥ 7.8 mmol/L was the cut-off for gestational diabetes. Women with gestational diabetes were referred for counselling and treatment and all women were followed until 6 weeks after delivery.FindingsOf 53 522 pregnant women screened, 6786 (12.7%) had gestational diabetes and were referred for nutritional therapy or medication; 836 (12.3%) of these women started medication. There was no significant difference in the prevalence of stillbirths between women with gestational diabetes (0.8%; 54/6786) and women without (0.7%; 338/46 736; P-value: 0.51). Of the women on treatment, 38 had abnormal blood glucose after delivery and continued with the medication. Two women with gestational diabetes died; they had other associated co-morbidities – pre-eclampsia and anaemia.ConclusionWe found a high prevalence of gestational diabetes, indicating the need for gestational diabetes screening and implementation of this project on a larger scale. Gestational diabetes screening at the community level is operationally feasible using the existing human resources and infrastructure of the reproductive health programmes.     

Analysis of COVID-19-associated rhino-orbital-cerebral mucormycosis patients in a tertiary care center in Northern India

Purpose:An unprecedented surge has been noted in rhino-orbital-Cerebral mucormycosis (ROCM) in times of current COVID-19 pandemic. The present prospective study aims to evaluate clinico-epidemiological profile, risk factors, management, and outcome of the cases of ROCM that presented to our tertiary care center during the study period from April to June 2021.Methods:All patients were subjected to complete history taking, ophthalmological examination, and imaging studies. The patients were staged and were treated with intravenous liposomal amphotericin B (AMB) and sino-nasal debridement of local necrotic tissue. Transcutaneous retrobulbar AMB (TRAMB), orbital decompression, and exenteration were instituted as indicated. All patients were followed up for a minimum of 6 months before arriving at the final outcome. Statistical analysis was performed.Results:A total of 49 patients presented during the study period, with a mean age of 42.2 years. The major risk factors included uncontrolled diabetes (89.8%), COVID-19 positivity (51.02%), and concurrent steroid use (38.77%). The most common presenting symptom was facial pain/swelling (43.65%), while the most common presenting sign was deterioration in vision (75.51%). Intravenous liposomal AMB was given to all patients along with sino-nasal debridement (85.71%), TRAMB (57.14%), orbital decompression (14.28%), and exenteration (12.24%). Overall, mortality at 6 months was 22.45% (11 patients). Age more than 60 years, intracranial extension, and HbA1c of more than 8.0% were observed to be statistically significant indicators of mortality.Conclusion:Early suspicion and timely diagnosis of mucormycosis at rhino-orbital stage is warranted in order to salvage life as well as visual function. TRAMB may prove as potentially favorable treatment modality in cases with limited orbital involvement.     

Diagnosis and Management of Atypical Hemolytic Uremic Syndrome In Children: Single Centre Experience

Atypical hemolytic uremic syndrome (aHUS) although rare is the commonest cause of acute renal failure (ARF) in children and has poor prognosis. We present single centre experience of aHUS. Thirty six children (29 males, 7 females) with mean age, 7.9 years presented with ARF, 2 children also had tonic–clonic type convulsions. Their hematology examination revealed hemolytic anemia with s. creatinine (SCr), 5.54 mg/dl. Acute HUS was observed in 75 %, acute on chronic HUS in 19.4 % and patchy cortical necrosis (PCN) in 5.6 % biopsies. Mean 5.4 plasma exchanges (PE) were carried out. Supportive management of anti-hypertensives and prednisone was also given. Recovery end points were establishment of urine output, improvement of SCr and hematological profile. Hematology and renal function profile improved variably in all children, 5.6 % died, relapse was observed in 80.5 % over mean 70 days; 13.9 % children are doing well over mean follow-up of 268.8 days. Thus poor prognosis was observed in 86.1 % children. Children with acute on chronic HUS and PCN did not recover. Six children who recovered had acute HUS. aHUS in Indian children occurs at an older age of around 8 years and chronic/irreversible changes on histopathology examination are harbingers of poor prognosis. PE is life-saving however further research for developing strategies to improve long-term survival is needed.     

Pulmonary valve preservation in Tetralogy of Fallot with a mildly hypoplastic annulus-should we do it?

Background  

The decision to preserve the pulmonary valve during intracardiac repair of Tetralogy of Fallot [TOF] is traditionally based on the intra-operative measurement of pulmonary annulus by a Hegar dilator as per Rowlatt’s table. We sought to evaluate if there can be flexibility in not using a transannular patch repair in Indian population with mildly hypoplastic pulmonary annulus.