Identification of protein Salpha gene mutations including four novel mutations in eight unrelated patients with protein S deficiency

Eight distinct and potentially causative mutations were identified in eight unrelated Japanese patients with protein S (PS) deficiency, by direct DNA sequencing of the protein Salpha (PSalpha) gene-specific polymerase chain reaction products of all 15 exons and exon/intron boundaries. There were five missense mutations, including two novel mutations (Cys80Tyr and Arg314His), and three showed a major impact on the expected gene products: novel mutations of a 5-bp deletion (delCTCTG887:Cys206Stop) and a nonsense mutation (Glu208Stop), as well as a previously reported splice site (exon 10 +5 A-->G) mutation. One of the patients showed compound heterozygosity for delCTCTG887 and 732A-->G. Investigation for the cosegregation state of these two mutations with PS deficiency in the patient's family suggested that the delCTCTG887 mutation was responsible for the abnormal phenotype and that the 732A-->G (Lys155Glu) mutation did not appear to play a key role. However, we also identified the same 732A-->G (Lys155Glu) mutation in an unrelated patient with apparent PS deficiency with severe pulmonary embolism, and found that this mutation seemed to cosegregate with a PS-deficient state in her family members. These data implied that unknown factor(s) other than the 732A-->G mutation itself might influence phenotypic expression of PS status in different individuals.     

Protocol digest of randomized phase II study of modified FOLFIRINOX versus gemcitabine plus nab-paclitaxel combination therapy for locally advanced pancreatic cancer: Japan clinical oncology group study (JCOG1407)

Gemcitabine is one of the standard treatments for locally advanced pancreatic cancer. Recent studies on metastatic pancreatic cancer have shown that combination chemotherapy with oxaliplatin, irinotecan, fluorouracil, and leucovorin (FOLFIRINOX) and gemcitabine plus nab-paclitaxel (GnP) prolonged the overall survival compared with gemcitabine alone. To select the most promising chemotherapy, a randomized phase II selection design trial was started in July 2016 to compare between modified FOLFIRINOX and GnP for patients with locally advanced pancreatic cancer. A total of 124 patients will be enrolled from 36 Japanese institutions within 2.5 years. The primary endpoint is the proportion of 1-year overall survival, and secondary endpoints are progression-free survival, distant metastasis-free survival, response rate in patients with target lesions, CA19-9 response, adverse events, treatment-related death, early death, grade 4 non-hematological toxicity, and dose intensity. This trial has been registered with the UMIN Clinical Trials Registry [http://www.umin.ac.jp/ctr/index.htm], and the registration number is UMIN000023143.     

Factors responsible for pathogenicity in chickens of a low-pathogenic H7N7 avian influenza virus isolated from a feral duck

Highly pathogenic avian influenza viruses have poly-basic amino acid sequences at the cleavage site in their hemagglutinin (HA). Although this poly-basic region is a prerequisite factor for pathogenicity in chickens, not much is known about additional factors responsible for the acquisition of pathogenicity of the duck influenza virus in chickens. Here, we introduced multiple basic amino acid residues into the HA cleavage site of the A/duck/Hokkaido/Vac-2/2004 (H7N7) strain of avian influenza virus, which has low pathogenicity in chickens; the resultant Vac2sub-P0 strain was not intravenously pathogenic in chickens. In contrast, the Vac2sub-P3 strain, which was recovered from three consecutive passages of Vac2sub-P0 in chicks, was intravenously pathogenic in chickens. Six amino acid substitutions were identified by comparison of the Vac2sub-P3 and Vac2sub-P0 genomic sequences: Lys123Glu in PB2, Asn16Asp in PB1, Glu227Gly and Ile388Thr in HA, Gly228Arg in M1, and Leu46Pro in M2. The results of intravenous inoculations of chickens with recombinant virus indicated that all six amino acid substitutions were required to varying degrees for Vac2sub-P3 pathogenicity, with Glu227Gly and Ile388Thr in HA being particularly essential. These results reveal the roles of additional viral factors in the acquisition of pathogenicity in addition to the previously characterized role of the poly-basic amino acid sequences at the HA cleavage site.     

Monoclonal and polyclonal immunoglobulin G deposits on tubular basement membranes of native and pretransplant kidneys: A retrospective study

Monoclonal tubular basement membrane immune deposits (TBMID) are associated with progression of interstitial injury in renal allograft. However, the significance of monoclonal and polyclonal TBMID in the native kidney remains unclear. We retrospectively analyzed 1894 native kidney biopsies and 1724 zero-hour biopsies performed between 2008 and 2018 in our institution. The rate of immunoglobulin G (IgG) TBMID was found to be 8.4% among native kidney biopsies and 0.4% among zero-hour biopsies. Polyclonal TBMID is common in IgG4-related tubulointerstitial nephritis (37.5%), diabetic nephropathy (31.3%) and lupus nephritis (25.5%). Monoclonal IgG TBMID was identified in seven cases, including three zero-hour biopsies. The combination of IgG1κ was observed in two cases, IgG1λ in three, and IgG2κ in two. Electron microscopy revealed powdery electron-dense deposits in all cases. Monoclonal gammopathy of undetermined significance was diagnosed in one case. Although one patient with focal segmental glomerulosclerosis developed renal failure, all others exhibited stable renal function. Monoclonal IgG TBMID in the native kidney is not associated with renal prognosis. However, this may be an interesting immunopathological finding that would help clarify the pathogenesis of TBM immune deposits. Further study for both monoclonal and polyclonal TBMID is required in the future.     

Antibacterial properties of main-chain cationic polymers prepared through amine–epoxy ‘Click’ polymerization

Poly(β-hydroxyl amine)s are prepared through an amine–epoxy ‘click’ polymerization process in water under ambient conditions. These materials could be subjected to a post-polymerization protonation/alkylation reaction at the nitrogen atom to yield quaternary ammonium salts in the polymer backbone. The antimicrobial activities indicated that polymers carrying butyl chains at the nitrogen atom are effective towards Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), as only 10–20 μg mL⁻¹ polymer concentrations are required to inhibit the bacterial growth by >90%. One of the candidates was also found to be effective towards Mycobacterium smegmatis (M. smegmatis) – a model organism to develop drugs against rapidly spreading tuberculosis (TB) infections. The hemolysis assay indicated that a majority of antimicrobial agents did not disrupt red blood cell membranes. The mechanistic studies suggested that cell wall disruption by the cationic polymers was the likely cause of bacterial death.

Poly(β-hydroxyl amine)s are shown to be potent antibacterial materials.

The concept of ‘click’ chemistry, as introduced by Sharpless and coworkers, refers to high efficiency, regioselective, modular, and atom-economic chemical reactions with a high degree of orthogonality and simple purification protocols.¹ Besides the preparation of small molecules, such reactions can also be applied as a polymerization process. To describe this variation, Tang and coworkers coined the term ‘click’ polymerization.^2–5 In this context, the reaction between the amine and epoxide functionalities is known to proceed under ambient and aqueous conditions to afford poly(β-hydroxyl amine)s.^6–9 The amine–epoxy reaction is catalyst-free and generates no by-product.^10,11 Therefore, a post-polymerization purification protocol is not necessary and the polymers can be produced in a quantitative yield. The polymers also have the advantage of carrying a tertiary amine site in the backbone. Paulusse and coworkers have shown that such amines have a large (up to 64%) buffering capacity in the pH range of 7.4–5.1. They harnessed this property to form complexes of the cationic polymers with anionic plasmid DNA useful in gene delivery applications.¹² Inspired by this study, we hypothesized that such main-chain amine groups could be employed in interacting with the anionic bacterial cell membrane to design antimicrobial polymers.^13–18 The avenue for alkylation at the nitrogen atoms also offers an opportunity for preparing quaternary ammonium analogues and comparing their properties with the tertiary amine polymers.¹⁹ To investigate this, we prepared a new family of poly(β-hydroxyl amine)s differing in their chemical composition and studied their antibacterial and hemolytic properties.The polymer synthesis follows a simple sequence (Scheme 1). Initially, commercially available primary amine and di-epoxide monomers are stirred in water for 48 hours under ambient conditions. This procedure leads to the formation of poly(β-hydroxyl amine)s polymer chains, which can be isolated through a simple freeze-drying procedure (Fig. 1 and S1–S27^†). Typically, the molecular weights of these polymers range from 1–6 kDa with dispersity (M_w/M_n) in the range of 1.2–1.8. A post-polymerization treatment with an aqueous hydrochloric acid solution leads to protonation of the backbone amines. Alternatively, alkyl iodide under silver tetrafluoroborate catalysis can yield alkylation at the nitrogen atom. The quaternization of the nitrogen atoms can be observed with the help of ¹H-NMR spectroscopy (Fig. S1–S19^†). The methylene groups adjacent to the nitrogen atoms in the precursor polymers can be seen at 2.6 ppm. Upon quaternization, these signals shift downfield by 1 ppm due to electron-deficient nature of the positively charged nitrogen atom. In all cases, these signals moved completely and a good area integration ratio could be observed between the side-chain protons and main-chain protons indicating a quantitative transformation of the tertiary amine atoms to quaternary amines in the polymer backbone.Open in a separate windowScheme 1Synthesis and post-polymerization modification of poly(β-hydroxyl amine)s.Open in a separate windowFig. 1Chemical structure of the polymers. The polymer names indicate the linker (R) in the diglycidyl ether monomers and the substituent(s) (R′/R′′) on the nitrogen atom. R = PEG (polyethylene glycol, m = 8–9), PPO (polypropylene oxide, m = 4–5), EO (ethylene oxide), BD (butanediol). R′ = Pr (C₃H₇), Bu (C₄H₉), Pen (C₅H₁₁), Hex (C₆H₁₃), Hep (C₇H₁₅). R′′ = Me (CH₃), Et (C₂H₅), Pr (C₃H₇), Bu (C₄H₉).The antimicrobial activity of the synthesized polymers was investigated by the broth microdilution method to determine the minimum inhibitory concentration (MIC_>90) against E. coli, S. aureus, and M. smegmatis bacterial species (21Antimicrobial and hemolytic properties of poly(β-hydroxyl amine)s

Liposome‐Encapsulated Hemoglobin Accelerates Bronchial Healing After Pneumonectomy in the Rat With or Without Preoperative Radiotherapy

Liposome‐encapsulated hemoglobin (LEH) has been reported to accelerate wound healing in the stomach and skin in an experimental setting. LEH was tested in bronchial anastomotic healing after radiation and pneumonectomy in the rat. Sprague‐Dawley rats (n = 61) received preoperative radiation (20 Gy) to the chest and underwent left pneumonectomy with bronchial stump closure using the Sweet method 4 days later, when they were randomized to receive intravenous infusion of LEH with high O₂ affinity (P₅₀O₂ = 17 mm Hg, 10 mL/kg, n = 32) or saline (n = 29). Additional rats (n = 18) were treated in the same way without preoperative radiation. Bronchial anastomotic healing was evaluated 2 days after surgery by determining the bursting pressure and infiltration of neutrophils, monocytes, and macrophages. Bronchial bursting pressure was elevated in the rats receiving LEH both in the unirradiated group (LEH 212 ± 78 vs. saline 135 ± 63 mm Hg, P < 0.05) and in rats with preoperative radiation (LEH 162 ± 48 vs. saline 116 ± 56 mm Hg, P < 0.01). Moreover, the percentage of rats with bursting pressure <100 mm Hg tended to be smaller in the unirradiated group (LEH 1/9 [11.1%] vs. saline 4/9 [44.4%], NS) and was significantly reduced in irradiated animals (LEH 3/32 [9.4%] vs. saline 11/29 [38%], P < 0.05). There were no morphological differences except for macrophage infiltration to the anastomotic area, which was significantly prominent in the LEH‐treated rats (P < 0.05) regardless of the presence or absence of preoperative irradiation (IR). The results suggest that LEH with high O₂ affinity may improve mechanical strength and morphological findings in bronchial anastomosis in rats regardless of the presence or absence of preoperative IR. The irradiated rats later treated with LEH had equivalent or better bronchial healing than that of saline‐treated naïve animals undergoing pneumonectomy alone.     

Development of gelatin flakes, a new type of anti-adhesive material: a preliminary study of in vivo rat adhesion models

To overcome the problems associated with sheet- or film-type anti-adhesive materials, we developed a new type of anti-adhesive material, gelatin flakes. We made two types of gelatin flakes with or without thermal cross-linking, and preliminarily examined their basic properties and the anti-adhesive efficacy using a rodent adhesion model. Both types of the gelatin flakes rapidly turned into gel and tightly attached the injured surfaces, absorbing the moisture and blood, when applied onto the abraded sites of rats. In addition, these flakes could be sprayed into the desired area by compressed air through a device with a long, thin tube, which could be used in laparoscopic surgery. The anti-adhesive effects of both types of gelatin flakes were similar, and both types were significantly superior compared to the non-treated group. Although further investigations are necessary, the gelatin flakes have unique and useful properties and satisfactory anti-adhesive effects, which indicate that they may be applicable in laparoscopic surgery.     

First Outbreak of an H5N8 Highly Pathogenic Avian Influenza Virus on a Chicken Farm in Japan in 2020

On 5 November 2020, a confirmed outbreak due to an H5N8 highly pathogenic avian influenza virus (HPAIV) occurred at an egg-hen farm in Kagawa prefecture (western Japan). This virus, A/chicken/Kagawa/11C/2020 (Kagawa11C2020), was the first HPAI poultry isolate in Japan in 2020 and had multiple basic amino acids—a motif conferring high pathogenicity to chickens—at the hemagglutinin cleavage site. Mortality of chickens was 100% through intravenous inoculation tests performed according to World Organization for Animal Health criteria. Phylogenetic analysis showed that the hemagglutinin of Kagawa11C2020 belongs to clade 2.3.4.4B of the H5 Goose/Guangdong lineage and clusters with H5N8 HPAIVs isolated from wild bird feces collected in Hokkaido (Japan) and Korea in October 2020. These H5N8 HPAIVs are closely related to H5N8 HPAIVs isolated in European countries during the winter of 2019–2020. Intranasal inoculation of chickens with 10⁶ fifty-percent egg infectious doses of Kagawa11C2020 revealed that the 50% chicken lethal dose was 10^4.63 and the mean time to death was 134.4 h. All infected chickens demonstrated viral shedding beginning on 2 dpi—before clinical signs were observed. These results suggest that affected chickens could transmit Kagawa11C2020 to surrounding chickens in the absence of clinical signs for several days before they died.     

Severe Hepatic Injury Caused by Imatinib Mesylate Administered for the Treatment of Chronic Myeloid Leukemia and the Efficacy of Prednisolone for its Management

Imatinib mesylate is a specific inhibitor of BCR-ABL tyrosine kinase, which is now widely used for the treatment of chronic myeloid leukemia (CML) with a high efficacy. Although severe hepatic injury caused by imatinib mesylate is rare, such a side effect may force patients to discontinue taking imatinib mesylate. In the present paper, we report on the case of a 51-year-old woman with CML who experienced hepatic injury with severe hyperbilirubinemia caused by imatinib mesylate. The findings from a liver biopsy specimen and her clinical course suggested the hepatic injury to presumably have been caused by an allergic mechanism. The co-administration of prednisolone was thus tried, and she has been able to continue imatinib mesylate administration without any liver dysfunction and finally was able to obtain a complete cytogenetic response.We therefore recommend that prednisolone should be tried when severe hepatic injury caused by imatinib mesylate is observed, since it might enable such patients to continue imatinib mesylate treatment and thereby improve the prognosis in such cases.     

Fate of full‐house immunofluorescence staining in renal allograft: A case report

Here, we report the case of a patient with renal allograft with full‐house immunofluorescence staining in the zero‐hour biopsy. Full‐house immunofluorescence staining is a well‐known characteristic of lupus nephritis. Previous studies have reported patients with full‐house immunofluorescence staining, but without other symptoms or serological findings; this condition is referred to as full‐house nephropathy. We identified only one case out of 2203 zero‐hour biopsies over 13 years. Zero‐hour biopsy presented no glomerular changes but showed full‐house immunofluorescence staining. Electron microscopy revealed a nonorganized electron‐dense deposit mainly in the mesangial lesion. Systemic lupus erythematosus (SLE)‐associated antibodies were negative, and complement deficiency was not observed in the donor patients. Deposition of immunoglobulin and complement levels markedly decreased within 1–3 years post transplantation. Neither donor nor recipient developed clinical or biological features of SLE; they showed good renal prognosis.