The PE-PPE Family of Mycobacterium tuberculosis: Proteins in Disguise.

Mycobacterium tuberculosis has thrived in parallel with humans for millennia, and despite our efforts, M. tuberculosis continues to plague us, currently infecting a third of the world's population. The success of M. tuberculosis has recently been attributed, in part, to the PE-PPE family; a unique collection of 168 proteins fundamentally involved in the pathogenesis of M. tuberculosis. The PE-PPE family proteins have been at the forefront of intense research efforts since their discovery in 1998 and whilst our knowledge and understanding has significantly advanced over the last two decades, many important questions remain to be elucidated. This review consolidates and examines the vast body of existing literature regarding the PE-PPE family proteins, with respect to the latest developments in elucidating their evolution, structure, subcellular localisation, function, and immunogenicity. This review also highlights significant inconsistencies and contradictions within the field. Additionally, possible explanations for these knowledge gaps are explored. Lastly, this review poses many important questions, which need to be addressed to complete our understanding of the PE-PPE family, as well as highlighting the challenges associated with studying this enigmatic family of proteins. Further research into the PE-PPE family, together with technological advancements in genomics and proteomics, will undoubtedly improve our understanding of the pathogenesis of M. tuberculosis, as well as identify key targets/candidates for the development of novel drugs, diagnostics, and vaccines.

Authorship diversity in Gastroenterology-related Cochrane systematic reviews: Inequities in global representation.

The need for promoting diversity and equitable authorship representation in academics faces increasing recognition, with some articles pointing out the lack of diversity in specific fields. Currently, there are no such articles scrutinizing the author diversity in the field of Gastroenterology. Cochrane systematic reviews are perceived worldwide to be amongst the highest quality of evidence available, thereby its conclusions often impact policy and practice globally. However, little is known about the current state of authorship diversity in Gastroenterology-related Cochrane reviews.

Two Uncommon Presentations of COVID-19-Associated Mucormycosis.

Mucormycosis is a rare opportunistic fungal infection commonly affecting immunocompromised individuals. There has been a surge in the number of these cases during the second wave of the Coronavirus Disease 2019 (COVID-19) in India. Mucormycosis has been reported to occur concurrently or a few weeks post-recovery from COVID-19. There have been multiple case reports/case series of rhino-orbital mucormycosis in India as a complication in COVID-19 pneumonia. We report two unique presentations of COVID-19-associated mucormycosis (CAM) in patients recently recovered from COVID-19. The first patient is an uncontrolled diabetic with Fournier's gangrene at presentation, which on further evaluation, showed features of mucormycosis. The second one is a case of uncontrolled diabetes with a previous COVID-19 infection presenting with pulmonary mucormycosis and aspergillosis, complicated by a gastropleural fistula. While liposomal amphotericin B (L-ampB) was started for both patients, they significantly deteriorated during their course of hospital stay due to the severity of the disease.

Are blood groups related to the distribution and severity of COVID-19? A cross-sectional study in a tertiary care hospital in South India.

Background: Blood groups are inherited traits that affect the susceptibility/severity of a disease. A clear relationship between coronavirus disease 2019 (COVID-19) and ABO blood groups is yet to be established in the Indian population. This study aimed to demonstrate an association of the distribution and severity of COVID-19 with ABO blood groups. Methods: A cross-sectional study was conducted after obtaining ethics approval (IEC 207/20) among hospitalized patients using in-patient records and analyzed on SPSS-25. Chi-square tests were used for the analysis of categorical data and independent sample t-test/Mann-Whitney U tests were used for continuous data. Results: The B blood group had the highest prevalence among COVID-19-positive patients. The AB blood group was significantly associated with acute respiratory distress syndrome (ARDS) (p = 0.03), sepsis (p = 0.02), and septic shock (p = 0.02). The O blood group was associated with significantly lower rates of lymphopenia and leucocytosis. However, no significant clinical association was seen in the O blood group. Conclusion: This study has demonstrated that blood groups have a similar distribution among patients hospitalized with COVID-19 in the South Indian population. Additionally, it preludes to a possible association between the AB blood group and ARDS, sepsis, and septic shock. Further studies having a larger representation of AB blood groups, especially in patients hospitalized for critical COVID-19, with adjustment for possible covariates, are warranted to provide a reliable estimate of the risk in the South Indian population.

Reversal of portal gastropathy with partial internal biliary diversion in a case of progressive familial intrahepatic cholestasis.

Progressive intrahepatic cholestasis is a rare, genetic disorder causing bile acid secretion or transport defects. It can result in intrahepatic cholestasis that can progress to end-stage liver disease. Diagnosis is made using a combination of clinical and biochemical approaches. Genetic testing is currently the gold standard for investigation. We report a case of an 18-month-old male child with cholestatic pattern of jaundice from 16 months of life, which was associated with features suggestive of portal gastropathy. Detailed workup led to the diagnosis of progressive intrahepatic cholestasis (type 2). Early diagnosis prevented the need for liver transplant, and the child underwent surgical treatment with partial internal biliary diversion. Portal gastropathy and disease progression dramatically changed with corrective surgery. The patient was symptom-free at 10-week follow-up. Detecting this rare genetic disorder early has very good therapeutic implications from the patient's perspective and their morbidity and mortality profile; if untreated, it has a high propensity to progress to end-stage liver disease. The requirement of surgical interventions and liver transplantation is individualized on a case-to-case basis. An early diagnosis and initiation of treatment can prevent the need for a liver transplant as shown in the present case.

Development of Novel Glucocorticoids for Use in Antibody-Drug Conjugates for the Treatment of Inflammatory Diseases.

Glucocorticoids (GCs) are widely used to treat a variety of autoimmune and inflammatory diseases; however, systemic delivery of GCs is associated with side effects that affect essentially every organ system, reflecting the nearly ubiquitous expression of the glucocorticoid receptor (GR). Targeted delivery of GCs to diseased tissues using antibody-glucocorticoid conjugates (GC-ADCs) offers a therapeutic alternative to overcome these adverse effects. Herein, we describe novel classes of GCs that exhibited greater potency than dexamethasone and budesonide, a 100-fold selectivity toward the GR over other nuclear receptors, and no in vitro safety liability in pharmacology assays (hERG, AMES) and that demonstrated a substantial reduction in tumor necrosis factor-α (TNF-α) release in mice challenged with lipopolysaccharide (LPS). The site-specific conjugated GC-ADCs via cathepsin-cleavable linkers were highly stable in plasma and specifically released GCs in antigen-positive cells, suggesting that these novel GCs can serve as ADC payloads to treat autoimmune and inflammatory diseases.

Pseudopapillary Tumor of the Pancreas: A Rare Cause of Extrahepatic Portal Hypertension.

A solid pseudopapillary tumor (SPT) of the pancreas is an uncommon neoplasm, characterized by a well-encapsulated mass, with low malignant potential. It occurs predominantly in young females. We present a case of SPT of the pancreas which presented with sinistral portal hypertension. Despite characteristic radiological findings due to its rarity, it may be missed to more common conditions like peptic ulcer disease. Delayed diagnosis can lead to complications like portal hypertension. To the best of our knowledge, in existing medical literature, SPT of the pancreas in males has rarely been described. In our case, we found that the tumor was causing extrahepatic portal hypertension which is also a very unique presentation of this tumor. Due to its vague clinical manifestations, definitive diagnosis is often a challenge hence requiring prompt investigations.

Information Formulation of the UDU Kalman Filter.

A new information formulation of the Kalman filter is presented where the information matrix is parameterized as the product of an upper triangular matrix, a diagonal matrix, and the transpose of the triangular matrix (UDU factorization). The UDU factorization of the Kalman filter is known for its numerical stability, this work extends the technique to the information filter. A distinct characteristic of the new algorithm is that measurements can be processed as vectors, while the classic UDU factorization requires scalar measurement processing, i.e. a diagonal measurement noise covariance matrix.

Preclinical Activity of the Novel Anti-Prolactin Receptor (PRLR) Antibody-Drug Conjugate REGN2878-DM1 in PRLR-Positive Breast Cancers.

The Prolactin Receptor (PRLR) is a type 1 cytokine receptor that is expressed in a subset of breast cancers and may contribute to its pathogenesis. It is relatively overexpressed in approximately 25% of human breast tumors while expressed at low levels in some normal human tissues including the mammary gland. We developed an anti-PRLR antibody-drug conjugate (ADC), to target PRLR-positive breast cancer. REGN2878-DM1 is comprised of a fully human high-affinity function-blocking anti-PRLR IgG1 antibody (REGN2878) conjugated via a noncleavable SMCC linker to the cytotoxic maytansine derivative DM1. Both unconjugated REGN2878 and conjugated REGN2878-DM1 block PRL-mediated activation in vitro and are rapidly internalized into lysosomes. REGN2878-DM1 induces potent cell-cycle arrest and cytotoxicity in PRLR-expressing tumor cell lines. In vivo, REGN2878-DM1 demonstrated significant antigen-specific antitumor activity against breast cancer xenograft models. In addition, REGN2878-DM1 showed additive activity when combined with the antiestrogen agent fulvestrant. These results illustrate promising antitumor activity against PRLR-positive breast cancer xenografts and support the evaluation of anti-PRLR ADCs as potential therapeutic agents in breast cancer. Mol Cancer Ther; 16(7); 1299-311. ©2017 AACR.

PET of tumors expressing gastrin-releasing peptide receptor with an 18F-labeled bombesin analog.

UNLABELLED: The gastrin-releasing peptide receptor (GRPR) is overexpressed in human prostate cancer. Bombesin (BBN) is a neurotransmitter of 14 amino acids and binds with selectivity and with high affinity to GRPRs. We have synthesized a NOTA-conjugated bombesin derivative, NOTA-8-Aoc-BBN(7-14)NH(2), to label this analog with (18)F using the new Al(18)F method. In this study, the GRPR-targeting potential of (18)F-labeled NOTA-8-Aoc-BBN(7-14)NH(2) was studied using (68)Ga-NOTA-8-Aoc-BBN(7-14)NH(2) as a reference. METHODS: The NOTA-conjugated bombesin analog was synthesized and radiolabeled with (68)Ga or (18)F. For (18)F labeling, we used our new 1-pot, 1-step method. The labeled product was purified by reversed-phase high-performance liquid chromatography. The log P values of the radiotracers were determined. The tumor-targeting characteristics of the compounds were assessed in mice with subcutaneously growing PC-3 xenografts. GRPR-binding specificity was studied by coinjection of an excess of unlabeled NOTA-8-Aoc-BBN(7-14)NH(2). Small-animal PET/CT images were acquired. RESULTS: NOTA-8-Aoc-BBN(7-14)NH(2) could be efficiently labeled with (18)F or with (68)Ga. NOTA-8-Aoc-BBN(7-14)NH(2) was labeled with (18)F in a single step, with 50%-90% yield. Radiolabeling, including purification, was performed in 45 min and resulted in a specific activity of greater than 10 GBq/µmol. The log P values of (18)F- and (68)Ga-labeled NOTA-8-Aoc-BBN(7-14)NH(2) were -1.47 ± 0.05 and -1.98 ± 0.03, respectively. In mice, both radiolabeled compounds cleared rapidly from the blood (<0.07 percentage injected dose per gram at 1 h after injection), mainly via the kidneys. At 1 h after injection, the uptake of (18)F- and (68)Ga-labeled NOTA-8-Aoc-BBN(7-14)NH(2) in the PC-3 tumors was 2.15 ± 0.55 and 1.24 ± 0.26 percentage injected dose per gram, respectively. GRPR-binding specificity was demonstrated by reduced tumor uptake of radiolabeled NOTA-8-Aoc-BBN(7-14)NH(2) after coinjection of a 100-fold excess of unlabeled NOTA-8-Aoc-BBN(7-14)NH(2) peptide. The accumulation of (18)F-NOTA-8-Aoc-BBN(7-14)NH(2) in the subcutaneous PC-3 tumors could be visualized via small-animal PET. CONCLUSION: NOTA-8-Aoc-BBN(7-14)NH(2) could be labeled rapidly and efficiently with (18)F using a 1-pot, 1-step method. Radiolabeled NOTA-8-Aoc-BBN(7-14)NH(2) specifically accumulated in the GRPR-expressing PC-3 tumors and should be evaluated clinically.

New lyophilized kit for rapid radiofluorination of peptides.

Radiolabeling compounds with positron-emitting radionuclides often involves a time-consuming, customized process. Herein, we report a simple lyophilized kit formulation for labeling peptides with (18)F, based on the aluminum-fluoride procedure. The prototype kit contains IMP485, a NODA (1,4,7-triazacyclononane-1,4-diacetate)-MPAA (methyl phenylacetic acid)-di-HSG (histamine-succinyl-glycine) hapten-peptide, [NODA-MPAA-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH(2)], used for pretargeting, but we also examined a similar kit formulation for a somatostatin-binding peptide [IMP466, NOTA-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Throl] bearing a NOTA ligand to determine if the benefits of using a kit can be extended to other AlF-binding peptides. The NODA-MPAA ligand forms a single stable complex with (AlF)(2+) in high yields. In order to establish suitable conditions for a facile kit, the formulation was optimized for pH, peptide to Al(3+) ratio, bulking agent, radioprotectant, and the buffer. For optimal labeling, the kit was reconstituted with an aqueous solution of (18)F(-) and ethanol (1:1), heated at 100-110 °C for 15 min, and then simply and rapidly purified using one of two equally effective solid-phase extraction (SPE) methods. Al(18)F-IMP485 was isolated as a single isomer complex, in high yield (45-97%) and high specific activity (up to 223 GBq/µmol), within 20 min. The labeled product was stable in human serum at 37 °C for 4 h and in vivo, urine samples showed the intact product was eliminated. Tumor targeting of the Al(18)F-IMP485 in nude mice bearing human colon cancer xenografts pretargeted with an anti-CEACAM5 bispecific antibody showed very low uptake (0.06% ± 0.02 ID/g) in bone, further illustrating its stability. At 1 h, pretargeted animals had high Al(18)F-IMP485 tumor uptake (28.1% ± 4.5 ID/g), with ratios of 9 ± 4, 123 ± 38, 110 ± 43, and 120 ± 108 for kidney, liver, blood and bone, respectively. Tumor uptake remained high at 3 h postinjection, with increased tumor/nontumor ratios. The NOTA-somatostatin-binding peptide also was fluorinated with good yield and high specific activity in the same kit formulation. However, yields were somewhat lower than those achieved with IMP485 containing the NODA-MPAA ligand, likely reflecting this ligand's superior binding properties over the simple NOTA. These studies indicate that (18)F-labeled peptides can be reproducibly prepared as stable Al-F complexes with good radiochemical yield and high specific activity using a simple, one-step, lyophilized kit followed by a rapid purification by SPE that provides the (18)F-peptide ready for patient injection within 30 min.

The radiolabeling of proteins by the [18F]AlF method.

A new ([(18)F]AlF)(2+)-binding ligand that contains 1,4,7-triazacyclononane-1,4-diacetate (NODA) attached to a methyl phenylacetic acid group (MPA) was conjugated to N-(2-aminoethyl)maleimide (EM) to form NODA-MPAEM. The NODA-MPAEM was labeled with ([(18)F]AlF)(2+) at 105°C in 49-82% yield and conjugated at room temperature to an antibody Fab' fragment in 69-80% yield (total time ∼50min) and with retention of immunoreactivity. These data indicate that the rapid and simple [(18)F]AlF-labeling method can be easily adapted for preparing heat-sensitive compounds with (18)F quickly and in high yields.

Optimized labeling of NOTA-conjugated octreotide with F-18.

We recently reported a facile method based on the chelation of [(18)F]aluminum fluoride (Al(18)F) by NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid). Here, we present a further optimization of the (18)F labeling of NOTA-octreotide (IMP466). Octreotide was conjugated with the NOTA chelate and was labeled with (18)F in a two-step, one-pot method. The labeling procedure was optimized with regard to the labeling buffer, ionic strength, peptide concentration, and temperature. Radiochemical yield, specific activity, in vitro stability, and receptor affinity were determined. Biodistribution of (18)F-IMP466 was studied in AR42J tumor-bearing mice. In addition, microPET/CT images were acquired. IMP466 was labeled with Al(18)F in a single step with 97% yield in the presence of 80% (v/v) acetonitrile or ethanol. The labeled product was purified by HPLC to remove unlabeled peptide and unbound Al(18)F. The radiolabeling, including purification, was performed for 45 min. Specific activities of 48,000 GBq/mmol could be obtained. (18)F-IMP466 showed a high tumor uptake and excellent tumor-to-blood ratios at 2 h post-injection. In addition, the low bone uptake indicated that the Al(18)F-NOTA complex was stable in vivo. PET/CT scans revealed excellent tumor delineation and specific accumulation in the tumor. Uptake in receptor-negative organs was low. NOTA-octreotide could be labeled with (18)F in quantitative yields using a rapid two-step, one-pot, method. The compound was stable in vivo and showed rapid accretion in SSTR(2)-receptor-expressing AR42J tumors in nude mice. This method can be used to label other NOTA-conjugated compounds such as RGD peptides, GRPR-binding peptides, and Affibody molecules with (18)F.

High-yielding aqueous 18F-labeling of peptides via Al18F chelation.

The coordination chemistry of a new pentadentate bifunctional chelator (BFC), NODA-MPAA 1, containing the 1,4,7-triazacyclononane-1,4-diacetate (NODA) motif with a methylphenylacetic acid (MPAA) backbone, and its ability to form stable Al(18)F chelates were investigated. The organofluoroaluminates were easily accessible from the reaction of 1 and AlF(3). X-ray diffraction studies revealed aluminum at the center of a slightly distorted octahedron, with fluorine occupying one of the axial positions. The tert-butyl protected prochelator 7, which can be synthesized in one step, is useful for coupling to biomolecules on solid phase or in solution. High yield (55-89%) aqueous (18)F-labeling was achieved in 10-15 min with a tumor-targeting peptide 4 covalently linked to 1. Defluorination was not observed for at least 4 h in human serum at 37 °C. These results demonstrate the facile application of Al(18)F chelation using BFC 1 as a versatile labeling method for radiofluorinating other heat-stable peptides for positron emission imaging.

Synthesis and evaluation of the anti-inflammatory properties of selenium-derivatives of celecoxib.

Celecoxib is a selective cyclooxygenase (COX)-2 inhibitor used to treat inflammation, while selenium is known to down-regulate the transcription of COX-2 and other pro-inflammatory genes. To expand the anti-inflammatory property, wherein celecoxib could inhibit pro-inflammatory gene expression at extremely low doses, we incorporated selenium (Se) into two Se-derivatives of celecoxib, namely; selenocoxib-2 and selenocoxib-3. In vitro kinetic assays of the inhibition of purified human COX-2 activity by these compounds indicated that celecoxib and selenocoxib-3 had identical K(I) values of 2.3 and 2.4µM; while selenocoxib-2 had a lower K(I) of 0.72µM. Furthermore, selenocoxib-2 inhibited lipopolysaccharide-induced activation of NF-κB leading to the down-regulation of expression of COX-2, iNOS, and TNFα more effectively than selenocoxib-3 and celecoxib in RAW264.7 macrophages and murine bone marrow-derived macrophages. Studies with rat liver microsomes followed by UPLC-MS-MS analysis indicated the formation of selenenylsulfide conjugates of selenocoxib-2 with N-acetylcysteine. Selenocoxib-2 was found to release minor amounts of Se that was effectively inhibited by the CYP inhibitor, sulphaphenazole. While these studies suggest that selenocoxib-2, but not celecoxib and selenocoxib-3, targets upstream events in the NF-κB signaling axis, the ability to effectively suppress NF-κB activation independent of cellular selenoprotein synthesis opens possibilities for a new generation of COX-2 inhibitors with significant and broader anti-inflammatory potential.

Improved 18F labeling of peptides with a fluoride-aluminum-chelate complex.

We reported previously the feasibility to radiolabel peptides with fluorine-18 ((18)F) using a rapid one-pot method that first mixes (18)F(-) with Al(3+) and then binds the (Al(18)F)(2+) complex to a NOTA ligand on the peptide. In this report, we examined several new NOTA ligands and determined how temperature, reaction time, and reagent concentration affected the radiolabeling yield. Four structural variations of the NOTA ligand had isolated radiolabeling yields ranging from 5.8% to 87% under similar reaction conditions. All of the Al(18)F NOTA complexes were stable in vitro in human serum, and those that were tested in vivo also were stable. The radiolabeling reactions were performed at 100 degrees C, and the peptides could be labeled in as little as 5 min. The IMP467 peptide could be labeled up to 115 GBq/micromol (3100 Ci/mmol), with a total reaction and purification time of 30 min without chromatographic purification.

A novel method of 18F radiolabeling for PET.

UNLABELLED: Small biomolecules are typically radiolabeled with (18)F by binding it to a carbon atom, a process that usually is designed uniquely for each new molecule and requires several steps and hours to produce. We report a facile method wherein (18)F is first attached to aluminum as Al(18)F, which is then bound to a chelate attached to a peptide, forming a stable Al(18)F-chelate-peptide complex in an efficient 1-pot process. METHODS: For proof of principle, this method was applied to a peptide suitable for use in a bispecific antibody pretargeting method. A solution of AlCl(3).6H(2)O in a pH 4.0 sodium-acetate buffer was mixed with an aqueous solution of (18)F to form the Al(18)F complex. This was added to a solution of IMP 449 (NOTA-p-Bn-CS-d-Ala-d-Lys(HSG)-d-Tyr-d-Lys(HSG)-NH(2)) (NOTA-p-Bn-CS is made from S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid; HSG is histamine-succinyl-glycine) and heated to 100 degrees C for 15 min. In vitro and in vivo stability and targeting ability of the Al(18)F-IMP 449 were examined in nude mice bearing LS174T human colonic tumors pretargeted with an anti-CEACAM5 bispecific antibody (TF2). RESULTS: The radiolabeled peptide was produced in 5%-20% yield with an estimated specific activity of 18,500-48,100 GBq (500-1,300 Ci)/mmol. The Al(18)F-IMP 449 was stable for 4 h in serum in vitro, and in animals, activity isolated in the urine 30 min after injection was bound to the peptide. Nonchelated Al(18)F had higher tissue uptake, particularly in the bones, than the chelated Al(18)F-IMP 449, which cleared rapidly from the body by urinary excretion. Tumor uptake was 30-fold higher with TF2-pretargeted Al(18)F-IMP 449 than with the peptide alone. Dynamic PET showed tumor localization within 30 min and rapid and thorough clearance from the body. CONCLUSION: The ability to bind highly stable Al(18)F to metal-binding ligands is a promising new labeling method that should be applicable to a diverse array of molecules for PET.

Antibody conjugates of 7-ethyl-10-hydroxycamptothecin (SN-38) for targeted cancer chemotherapy.

CPT-11 is a clinically used cancer drug, and it is a prodrug of the potent topoisomerase I inhibitor, SN-38 (7-ethyl-10-hydroxycamptothecin). To bypass the need for the in vivo conversion of CPT-11 and increase the therapeutic index, bifunctional derivatives of SN-38 were prepared for use in antibody-based targeted therapy of cancer. The general synthetic scheme incorporated an acetylene-azide click cycloaddition step in the design, a short polyethylene glycol spacer for aqueous solubility, and a maleimide group for conjugation. Conjugates of a humanized anti-CEACAM5 monoclonal antibody, hMN-14, prepared using these SN-38 derivatives were evaluated in vitro for stability in buffer and human serum and for antigen-binding and cytotoxicity in a human colon adenocarcinoma cell line. Conjugates of hMN-14 and SN-38 derivatives 16 and 17 were found promising for further development.