[Surgical Experience with Three Cases of Open-window Thoracostomy for Chronic Empyema Under Local Anesthesia].

We may encounter patients with chronic empyema for whom open-window thoracostomy is unavoidable. However, patients with chronic empyema are sometimes at high-risk for surgery under general anesthesia. We, herein, present our surgical experience with three chronic empyema cases who underwent open-window thoracostomy under local anesthesia. Indications for open-window thoracostomy under local anesthesia were raised PaCO2 in Case 1, old age and poor performance status in Case 2, and a history of esophageal reconstruction and vocal cordoplasty in Case 3. All patients were well during the surgery. Case 1 developed type 2 respiratory failure postoperatively and had to be put on a ventilator, but finally recuperated. The sedatives used could have exacerbated raised PaCO2 in this patient, and careful selection of anesthetic agents is mandatory. Considering pain and stress that patients suffer during open-window thoracostomy under local anesthesia, case selection is necessary. Nevertheless, we believe that open-window thoracostomy under local anesthesia is an effective option for high-risk patients.

Acute leukemias in pregnant women: Results of a retrospective study at a local tertiary-care hospital in Japan.

Leukemia may rarely develop in a woman during pregnancy, posing clinical challenges to the patient, fetus, family, and medical staff managing malignancy and pregnancy. We retrospectively analyzed cases of pregnancy-associated leukemia consecutively diagnosed and treated at a local tertiary-care hospital in Nagano, Japan, over the past 20 years. Five cases were identified among 377,000 pregnancies in the area (one in every 75,000 pregnancies), all involving acute leukemia (three acute myelogenous leukemia [AML] and two acute lymphoblastic leukemia [ALL]). The cases were diagnosed in the first trimester (n = 1), second trimester (n = 3), or third trimester (n = 1). There were no apparent pregnancy-associated delays in diagnosing and treating the cases. Three patients underwent induction chemotherapy during pregnancy, two of whom eventually delivered healthy babies. One of the five patients chose abortion before chemotherapy initiation. Two cases showing high-risk features at the diagnosis (AML with an FLT3-ITD mutation [n = 1] and relapsed ALL [n = 1]) eventually died despite consolidative allogeneic hematopoietic stem cell transplantation. Our results suggested that patients with pregnancy-associated acute leukemia can be treated similarly to nonpregnant patients, although pregnancy imposes particular clinical challenges that should be resolved with multidisciplinary care.

The Molecular Basis of Heat-Stable Enterotoxin for Vaccine Development and Cancer Cell Detection.

Heat-stable enterotoxin (STa) produced by Enterotoxigenic E. coli is responsible for causing acute diarrhea in infants in developing countries. However, the chemical synthesis of STa peptides with the native conformation and the correct intra-molecular disulfide bonds is a major hurdle for vaccine development. To address this issue, we herein report on the design and preparation of STa analogs and a convenient chemical method for obtaining STa molecules with the correct conformation. To develop an STa vaccine, we focused on a structure in a type II ß-turn in the STa molecule and introduced a D-Lys residue as a conjugation site for carrier proteins. In addition, the -Glu-Leu- sequence in the STa molecule was replaced with a -Asp-Val- sequence to decrease the toxic activity of the peptide to make it more amenable for use in vaccinations. To solve several issues associated with the synthesis of STa, such as the formation of non-native disulfide isomers, the native disulfide pairings were regioselectively formed in a stepwise manner. A native form or topological isomer of the designed STa peptide, which possesses a right-handed or a left-handed spiral structure, respectively, were synthesized in high synthetic yields. The conformation of the synthetic STa peptide was also confirmed by CD and NMR spectroscopy. To further utilize the designed STa peptide, it was labeled with fluorescein for fluorescent detection, since recent studies have also focused on the use of STa for detecting cancer cells, such as Caco-2 and T84. The labeled STa peptide was able to specifically and efficiently detect 293T cells expressing the recombinant STa receptor (GC-C) protein and Caco-2 cells. The findings reported here provide an outline of the molecular basis for using STa for vaccine development and in the detection of cancer cells.

Structural dynamics of the N-terminal SH2 domain of PI3K in its free and CD28-bound states.

Protein conformational changes with fluctuations are fundamental aspects of protein-protein interactions (PPIs); understanding these motions is required for the rational design of PPI-regulating compounds. Src homology 2 (SH2) domains are commonly found in adapter proteins involved in signal transduction and specifically bind to consensus motifs of proteins containing phosphorylated tyrosine (pY). Here, we analysed the interaction between the N-terminal SH2 domain (nSH2) of the regulatory subunit in phosphoinositide 3-kinase (PI3K) and the cytoplasmic region of the T-cell co-receptor, CD28, using NMR and molecular dynamics (MD) simulations. First, we assigned the backbone signals of nSH2 on 1 H-15 N heteronuclear single quantum coherence spectra in the absence or presence of the CD28 phosphopeptide, SDpYMNMTPRRPG. Chemical shift perturbation experiments revealed allosteric changes at the BC loop and the C-terminal region of nSH2 upon CD28 binding. NMR relaxation experiments showed a conformational exchange associated with CD28 binding in these regions. The conformational stabilisation of the C-terminal region correlated with the regulation of PI3K catalytic function. Further, using 19 F- and 31 P-labelled CD28 phosphopeptide, we analysed the structural dynamics of CD28 and demonstrated that the aromatic ring of the pY residue fluctuated between multiple conformations upon nSH2 binding. Our MD simulations largely explained the NMR results and the structural dynamics of nSH2 and CD28 in both bound and unbound states. Notably, in addition to its major conformation, we detected a minor conformation of nSH2 in the CD28 bound state that may explain the allosteric conformational change in the BC loop.

Comparative analysis of humoral responses to BNT162b2 vaccine among patients with hematologic disorders and organ transplant recipients.

Vaccination against SARS-COV-2 is considered the most promising approach to curbing the pandemic. Patients with an immunocompromised state, such as those with hematological malignancies and organ transplantation recipients, are considered more susceptible to infection, but these at-risk patients were underrepresented in early clinical trials for vaccination. Although a growing body of studies suggests that the humoral response to COVID-19 vaccination in each of these at-risk groups of patients may be suboptimal in comparison to healthy controls, a clinical and strategic information for the further comparative analysis among these groups is not fully described. The humoral responses after two doses of BNT162b2 vaccination were evaluated in a total of 187 patients either with allogeneic hematopoietic transplantation, with renal transplantation, with anti-CD20 antibody therapy, or with anti-CD38 antibody therapy, and in 66 healthy controls. The early response at one to three months after vaccination was significantly inferior among patients with renal transplantation, patients with anti-CD20 antibody therapy, and patients with anti-CD38 antibody therapy in comparison to healthy control. But the patients with allogeneic hematopoietic transplantation showed early humoral response comparable to healthy control. The late response at 6 months after vaccination was still suboptimal among patients with renal transplantation and patients with anti-CD20 therapy. Among our patient group, renal transplant recipients had the lowest antibody titers after vaccination regardless of timing of vaccination. Patients who had received allogeneic hematopoietic transplantation attained a comparable serological response to the control group especially if they are vaccinated >300 days after transplantation, but the response was suboptimal if the vaccination was within 300 days after transplantation. Our results may provide policy makers with critical information for the further stratification of at-risk groups, helping contribute to a better allocation of resources, including additional booster vaccination.

Somatic mutations in acquired pure red cell aplasia.

Acquired pure red cell aplasia (PRCA) is a syndrome characterized by anemia and a marked reduction of erythroid progenitor cells with various etiologies. The 3 major subtypes of PRCA are idiopathic PRCA, large granular lymphocytic leukemia-associated PRCA and thymoma-associated PRCA, which are thought to be caused by a T-cell-mediated mechanism. In these 3 subtypes, an expansion of clonal cytotoxic T cells is often detected. In addition, those T cells recurrently harbor somatic mutations of STAT3, a gene coding one of the important signal transducers in the JAK/STAT system. Somatic mutations of clonal hematopoiesis (CH)-related genes, including epigenetic modifying genes, have also been reported, however, the data are still not mature enough upon which to draw conclusion, Somatic mutations of STAT3 and CH-related genes may be unique characteristics of acquired PRCA. However, their involvement in dyserythropoiesis or clinical relevance to the clinical course of those somatic mutations. Mutational landscapes, their involvements in dyserythropoiesis and clinical relevance in acquired PRCA remains unclear, and further investigation is needed.

Anti-COX-2 autoantibody is a novel biomarker of immune aplastic anemia.

In immune aplastic anemia (IAA), severe pancytopenia results from the immune-mediated destruction of hematopoietic stem cells. Several autoantibodies have been reported, but no clinically applicable autoantibody tests are available for IAA. We screened autoantibodies using a microarray containing >9000 proteins and validated the findings in a large international cohort of IAA patients (n = 405) and controls (n = 815). We identified a novel autoantibody that binds to the C-terminal end of cyclooxygenase 2 (COX-2, aCOX-2 Ab). In total, 37% of all adult IAA patients tested positive for aCOX-2 Ab, while only 1.7% of the controls were aCOX-2 Ab positive. Sporadic non-IAA aCOX-2 Ab positive cases were observed among patients with related bone marrow failure diseases, multiple sclerosis, and type I diabetes, whereas no aCOX-2 Ab seropositivity was detected in the healthy controls, in patients with non-autoinflammatory diseases or rheumatoid arthritis. In IAA, anti-COX-2 Ab positivity correlated with age and the HLA-DRB1*15:01 genotype. 83% of the >40 years old IAA patients with HLA-DRB1*15:01 were anti-COX-2 Ab positive, indicating an excellent sensitivity in this group. aCOX-2 Ab positive IAA patients also presented lower platelet counts. Our results suggest that aCOX-2 Ab defines a distinct subgroup of IAA and may serve as a valuable disease biomarker.

Single-cell characterization of leukemic and non-leukemic immune repertoires in CD8+ T-cell large granular lymphocytic leukemia.

T cell large granular lymphocytic leukemia (T-LGLL) is a rare lymphoproliferative disorder of mature, clonally expanded T cells, where somatic-activating STAT3 mutations are common. Although T-LGLL has been described as a chronic T cell response to an antigen, the function of the non-leukemic immune system in this response is largely uncharacterized. Here, by utilizing single-cell RNA and T cell receptor profiling (scRNA+TCRαß-seq), we show that irrespective of STAT3 mutation status, T-LGLL clonotypes are more cytotoxic and exhausted than healthy reactive clonotypes. In addition, T-LGLL clonotypes show more active cell communication than reactive clones with non-leukemic immune cells via costimulatory cell-cell interactions, monocyte-secreted proinflammatory cytokines, and T-LGLL-clone-secreted IFNγ. Besides the leukemic repertoire, the non-leukemic T cell repertoire in T-LGLL is also more mature, cytotoxic, and clonally restricted than in other cancers and autoimmune disorders. Finally, 72% of the leukemic T-LGLL clonotypes share T cell receptor similarities with their non-leukemic repertoire, linking the leukemic and non-leukemic repertoires together via possible common target antigens. Our results provide a rationale to prioritize therapies that target the entire immune repertoire and not only the T-LGLL clonotype.

T cell clonal expansion and STAT3 mutations: a characteristic feature of acquired chronic T cell-mediated pure red cell aplasia.

Acquired chronic pure red cell aplasia (PRCA) develops idiopathically or in association with other medical conditions, including T cell large granular lymphocytic leukemia (T-LGLL) and thymoma. T cell dysregulation is considered a cardinal pathogenesis of PRCA, but genetic-phenotypic associations in T cell abnormalities are largely unclear. We evaluated an extended cohort of 90 patients with acquired PRCA, including 26 with idiopathic, 36 with T-LGLL-associated and 15 with thymoma-associated PRCA, for their T cell immuno-phenotypes, clonalities and STAT3 mutations. TCR repertoire skewing of CD8+ T cells was detected in 37.5% of idiopathic, 66.7% of T-LGLL-associated and 25% of thymoma-associated PRCA patients, and restriction to Vß1 was most prominent (41%). Clonalities of TCRß or γ chain and STAT3 mutational status were statistically associated (P = 0.0398), and they were detected in all three subtypes. The overall response rate to cyclosporin A was 73.9%, without significant difference by subtypes nor STAT3 mutational status. The T cell dysregulations, such as TCR repertoire skewing with predominant Vß1 usage, clonality and STAT3 mutations, were frequently found across the subtypes, and the close associations between them suggest that these T cell derangements reflect a common pathophysiological mechanism among these PRCA subtypes.

Identification of novel STAT5B mutations and characterization of TCRß signatures in CD4+ T-cell large granular lymphocyte leukemia.

CD4+ T-cell large granular lymphocyte leukemia (T-LGLL) is a rare subtype of T-LGLL with unknown etiology. In this study, we molecularly characterized a cohort of patients (n = 35) by studying their T-cell receptor (TCR) repertoire and the presence of somatic STAT5B mutations. In addition to the previously described gain-of-function mutations (N642H, Y665F, Q706L, S715F), we discovered six novel STAT5B mutations (Q220H, E433K, T628S, P658R, P702A, and V712E). Multiple STAT5B mutations were present in 22% (5/23) of STAT5B mutated CD4+ T-LGLL cases, either coexisting in one clone or in distinct clones. Patients with STAT5B mutations had increased lymphocyte and LGL counts when compared to STAT5B wild-type patients. TCRß sequencing showed that, in addition to large LGL expansions, non-leukemic T cell repertoires were more clonal in CD4+ T-LGLL compared to healthy. Interestingly, 25% (15/59) of CD4+ T-LGLL clonotypes were found, albeit in much lower frequencies, in the non-leukemic CD4+ T cell repertoires of the CD4+ T-LGLL patients. Additionally, we further confirmed the previously reported clonal dominance of TRBV6-expressing clones in CD4+ T-LGLL. In conclusion, CD4+ T-LGLL patients have a typical TCR and mutation profile suggestive of aberrant antigen response underlying the disease.

Structural and spectroscopic characterization of CO inhibition of [NiFe]-hydrogenase from Citrobacter sp. S-77.

Hydrogenases catalyze the reversible oxidation of H2. Carbon monoxide (CO) is known to be a competitive inhibitor of O2-sensitive [NiFe]-hydrogenases. Although the activities of some O2-tolerant [NiFe]-hydrogenases are unaffected by CO, the partially O2-tolerant [NiFe]-hydrogenase from Citrobacter sp. S-77 (S77-HYB) is inhibited by CO. In this work, the CO-bound state of S77-HYB was characterized by activity assays, spectroscopic techniques and X-ray crystallography. Electron paramagnetic resonance spectroscopy showed a diamagnetic Ni2+ state, and Fourier-transform infrared spectroscopy revealed the stretching vibration of the exogenous CO ligand. The crystal structure determined at 1.77 Šresolution revealed that CO binds weakly to the nickel ion in the Ni-Fe active site of S77-HYB. These results suggest a positive correlation between O2 and CO tolerance in [NiFe]-hydrogenases.

Relative configuration of Cys-Pro ester peptides in thioester formation.

A peptide containing a cysteinyl prolyl ester (CPE) moiety at the C-terminus (CPE peptide) was transformed into a diketopiperazine (DKP) thioester via an intramolecular N-S acyl shift reaction and was then used for peptide ligation. The difference in reactivity between the CPE peptide stereoisomers was examined. In reactions of the CPE peptides that contained L-Cys-L-Pro or D-Cys-D-Pro, the desired DKP thioester was formed at the preceding amino acid residue. On the other hand, in reactions of the CPE peptides that contained D-Cys-L-Pro or L-Cys-D-Pro, a thiolactone was formed at the C-terminal prolyl ester, and the ligation occurred at the C-terminal Pro residue. Using this reaction, it was possible to efficiently prepare a cyclic peptide.

Structural dynamics of the chromo-shadow domain and chromodomain of HP1 bound to histone H3K9 methylated peptide, as measured by site-directed spin-labeling EPR spectroscopy.

The structural dynamics of the chromo-shadow domain (CSD) and chromodomain (CD) of human HP1 proteins essential for heterochromatin formation were investigated at the nanosecond and nanometer scales by site-directed spin labeling electron paramagnetic resonance and pulsed double resonance spectroscopy. Distance measurements showed that the spin-labeled CSD of human HP1α and HP1γ tightly dimerizes. Unlike CD-CD interaction observed in fission yeast HP1 in an inactivated state (Canzio et al., 2013), the two CDs of HP1α and HP1γ were spatially separated from each other, dynamically mobile, and ready for a Brownian search for H3K9-tri-methyl(me3) on histones. Complex formation of the CD with H3K9me3 slowed dynamics of the domain due to a decreased diffusion constant. CSD mobility was significantly (∼1.3-fold) lower in full-length HP1α than in HP1γ, suggesting that the immobilized conformation of human HP1α shows an auto-inactivated state. Differential properties of HP1α and HP1γ to form the inactive conformation could be relevant to its physiological role in the heterochromatin formation in a cell.

A novel germline GATA2 frameshift mutation with a premature stop codon in a family with congenital sensory hearing loss and myelodysplastic syndrome.

GATA2 is a zinc-finger transcription factor regulating early hematopoiesis and developmental processes. Heterozygous germline mutations in GATA2 underlie a pleiotropic autosomal dominant disorder, GATA2 deficiency syndrome. The wide spectrum of its clinical features involves familial predisposition to myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) and multiorgan dysfunction, including congenital sensorineural hearing loss (CSHL). We herein report a pedigree with a novel germline frameshift mutation presenting as CSHL and familial MDS. The proband was a 46-year-old man, and his daughter also presented with an identical set of clinical syndromes. Target DNA sequencing identified a novel eight-nucleotide duplicative insertion at exon 5 (NM_032638.4:c.1126_1133dup:p.Lys378Asnfs*12)　of the GATA2 gene. RT-PCR and subcloning analysis showed that the frameshift might result in a truncated mutation with an early stop codon without interfering with the predicted splice site. The predicted mutant protein had 388 amino acids and in silico analysis showed the variant was considered deleterious. This mutation was not detected in unaffected family members. Its deleterious effect is highly likely to have portended the familial MDS and CSHL in this pedigree. Genetic testing among suspected individuals may be warranted for adequate management, including timely transplantation.

Isolated splenic Mycobacterium tuberculosis complex infection in an immunocompetent individual with FDG-PET positive mass.

Tuberculosis, caused by Mycobacterium tuberculosis complex, is a leading cause of mortality in the world, and 15% of the patients may present with extrapulmonary diseases, including splenic lesion. However, isolated splenic infection with M. tuberculosis complex is very rare. A 19-year-old otherwise healthy woman presented with left flank pain, revealing FDG-avid nodules in the spleen. She did not have pulmonary lesions. Histopathology of splenectomized sample showed granuloma, and subsequent PCR revealed amplification of IS6110, a genetic sequence exclusively detected in M. tuberculosis complex. A wide range of differential diagnosis of isolated splenic lesion should include M. tuberculosis infection regardless of pulmonary involvement. An elective splenectomy may be mandatory in timely manner.

Chemical Digestion of the -Asp-Cys- Sequence for Preparation of Post-translationally Modified Proteins.

Numerous studies of native proteins have been reported on protein folding in this half century. Recently, post-translationally modified proteins are also focused on protein folding. However, it is still difficult to prepare such types of proteins because it requires not only the chemical but also the recombinant techniques. Native chemical ligation (NCL) is a powerful technique for producing target proteins when combined with recombinant techniques, such as expressed protein ligation (EPL). NCL basically requires an N-terminal peptide with a thioester and a C-terminal peptide which should possess a Cys residue at the N-terminus. Numerous efforts have been made to prepare N-terminal peptides carrying a thioester or a derivative thereof. However, a method for preparing C-terminal Cys-peptides with post-translational modifications has not been well developed, making it difficult to prepare such C-terminal Cys-peptides, except for chemical syntheses or enzymatic digestion. We report here on the development of a convenient technique that involves acid hydrolysis at the -Asp-Cys- sequence, to effectively obtain a C-terminal peptide fragment that can be used for any protein synthesis when combined with EPL, even under denatured conditions. Thus, this chemical digestion strategy permits the NCL strategy to be dramatically accelerated for protein syntheses in which post-translational modifications, such as glycosylation, phosphorylation, etc. are involved. In addition, this method should be useful to prepare the post-translationally modified proteins for protein folding.

Copper stabilizes antiparallel ß-sheet fibrils of the amyloid ß40 (Aß40)-Iowa variant.

Cerebral amyloid angiopathy (CAA) is a vascular disorder that primarily involves deposition of the 40-residue-long ß-amyloid peptide (Aß40) in and along small blood vessels of the brain. CAA is often associated with Alzheimer's disease (AD), which is characterized by amyloid plaques in the brain parenchyma enriched in the Aß42 peptide. Several recent studies have suggested a structural origin that underlies the differences between the vascular amyloid deposits in CAA and the parenchymal plaques in AD. We previously have found that amyloid fibrils in vascular amyloid contain antiparallel ß-sheet, whereas previous studies by other researchers have reported parallel ß-sheet in fibrils from parenchymal amyloid. Using X-ray fluorescence microscopy, here we found that copper strongly co-localizes with vascular amyloid in human sporadic CAA and familial Iowa-type CAA brains compared with control brain blood vessels lacking amyloid deposits. We show that binding of Cu(II) ions to antiparallel fibrils can block the conversion of these fibrils to the more stable parallel, in-register conformation and enhances their ability to serve as templates for seeded growth. These results provide an explanation for how thermodynamically less stable antiparallel fibrils may form amyloid in or on cerebral vessels by using Cu(II) as a structural cofactor.

Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation.

Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance.

Magnetic Resonance Imaging-negative, Rituximab-resistant Neurolymphomatosis as a Paradoxical Presentation of Relapsed Primary Adrenal Lymphoma.

Primary adrenal lymphoma (PAL) is rare and known to have a predilection for central nervous system (CNS) relapse. A 70-year-old man with a 2-year history of primary aldosteronism presented because of a fever. He was hypotensive, and his adrenal glands were unequivocally enlarged. PAL was diagnosed. Despite showing an initial response to immunochemotherapy, progressive paralysis ensued. Magnetic resonance imaging findings were negative, and rituximab was ineffective. His debilitated condition hindered further chemotherapy. A postmortem examination revealed lymphoma relapse in the systemic peripheral nerves. The sequential presentation of two rare lymphomas implies that PAL might have a predilection for not only the CNS but also peripheral nerves.