Antibody-like proteins that capture and neutralize SARS-CoV-2.

To combat severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and any unknown emerging pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or antibody-like proteins is of critical importance. We here report high-speed in vitro selection of multiple high-affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of monobodies against the SARS-CoV-2 spike protein were successfully procured within only 4 days. Furthermore, the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and exhibited a high neutralizing activity against SARS-CoV-2 infection (half-maximal inhibitory concentration, 0.5 nanomolar). High-speed in vitro selection of antibody-like proteins is a promising method for rapid development of a detection method for, and of a neutralizing protein against, a virus responsible for an ongoing, and possibly a future, pandemic.

Linker-free incorporation of carbohydrates into in vitro displayed macrocyclic peptides.

We report a strategy for efficient post-translational modification of a library of ribosomally-translated peptides by activation and elimination of cysteine to dehydroalanine then conjugate addition of a range of exogenous thiols, with an emphasis on carbohydrates. These reactions are selective for cysteine, and do not interfere with amplification of the nucleic acid component of an mRNA-displayed peptide. Furthermore, these reactions are shown to be compatible with two different macrocyclisation chemistries, and when applied to a peptide containing an N-terminal cysteine give a ketone that can be functionalised in an orthogonal manner. This new strategy can overcome a limitation of ribosomal translation, providing a means to incorporate untranslatable groups such as carbohydrates in amino acid side chains, and will allow for the ribosomal generation of glycopeptides, requiring only the introduction of a free thiol in the molecule to be incorporated. In combination with in vitro selection techniques, this strategy is envisaged to allow the discovery of biologically-active glycopeptides with a near-natural, but hydrolytically stable, thioglycosidic bond.

Impact of pre-treatment prostate tissue androgen content on the prediction of castration-resistant prostate cancer development in patients treated with primary androgen deprivation therapy.

Great advances in tissue androgen analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) have made it possible to evaluate the tissue androgen content from a single needle prostate biopsy specimen. In this study, we investigated if pre-treatment androgen content in prostate biopsy specimens could predict their response to primary androgen deprivation therapy (ADT) and future castration-resistant prostate cancer (CRPC). One-hundred and sixty-five prostate cancer patients who received primary ADT were enrolled. They had received multiple core prostate needle biopsy at diagnosis, and an additional one needle biopsy specimen was obtained for tissue androgen determination using LC-MS/MS. The patients' prostate specific antigen (PSA) values were periodically followed during the treatment and patients were determined to have CRPC when their PSA value increased continuously to 25% above the nadir and a 2.0 ng/mL increase. A significant correlation was found between PSA value decline velocity (PSA half-time) after ADT and pre-ADT tissue androgen content. Twenty-three patients were determined to have CRPC. These CRPC patients had a significantly high concentration of tissue T (p < 0.01) and low concentration of tissue 5α-dihydrotestosterone (DHT) (p < 0.01), resulting in a higher tissue T/DHT ratio (p < 0.001). A multivariate Cox proportional hazard model revealed the pre-ADT tissue T/DHT ratio and Gleason score as independent predictors for CRPC development. By using the two statistically significant variables, the relative risk of CRPC development could be calculated. The results of this study suggest that the evaluation of prostate androgen content in a single needle biopsy specimen may be useful to predict future CRPC development after primary ADT. Further studies are required for the clinical application of T/DHT ratio evaluation.

Pure laparoscopic hepatectomy for hepatocellular carcinoma patients with severe liver cirrhosis.

Hepatocellular carcinoma often arises in cirrhotic livers. Patients with severe liver cirrhosis who undergo hepatectomy often develop postoperative liver failure, even if the hepatectomy is limited. Here, we report six patients with severe liver cirrhosis (Child-Pugh B/C and indocyanine green retention rate at 15 min ≥ 40%) who underwent pure laparoscopic hepatectomy. Their perioperative course was favorable and comparable to that of other hepatocellular carcinoma patients with mild-moderate liver cirrhosis. In patients with severe liver cirrhosis, pure laparoscopic hepatectomy minimizes the disturbance in collateral blood and lymphatic flow caused by laparotomy and liver mobilization, as well as the mesenchymal injury caused by compression of the liver. It limits complications such as massive ascites, which can lead to severe postoperative liver failure. Good candidates for the procedure include patients with severe liver cirrhosis who have tumors on the liver surface and in whom adaptation to ablation therapy is difficult and/or who experience local recurrence after repeat treatments.

Budgets of major ionic species and nutrients on a dam reservoir in forested watershed.

To evaluate the role of a dam reservoir in the runoff of pollutant loadings from a forested watershed, the input-output budgets in the Ikuno dam reservoir had been investigated for eight years since 1996. The T-N, T-P, TOC and major ionic species in the bulk precipitation, stream water, and outflow were measured. The residence time calculated by using the data of the inflow and outflow was 0.3 year. The average precipitation was 1,772 mm during the investigation period (1996-2004). The direct deposition to water surface was less than one percentage to total loadings of nutrients and major ionic species. The ratios of output to input of TOC, TN, and TP were 1.04 to 1.42, and those of major ionic species were from 0.83 to 0.99 except for NO3(-), which was 1.12. However, the ratios of output to input of major ionic species except for NO3(-) at the Ikuno dam reservoir will be larger, and those of NO3(-), TOC, TN, and TP will be smaller, if we also include rain events. These results suggested that the dam reservoir played a role as a sink for pollutants in forested watershed, and that the pollutant loadings to downstream may decrease.

Application of an automatic sampling and measurement system to a mountainous stream investigation during rain events.

An automatic sampling and measurement system was developed to take water samples efficiently during rain events. The system consisted of an automatic sampler, a rain gauge and a data logger as well as sensors for pH, electrical conductivity (EC), and both water and air temperature. The system was tested in a stream in a forested watershed (5.8 km2) located in the middle of Hyogo prefecture, Japan. The sampling program has been improved gradually. For several rain events of 30 to 157 mm, most water samples were in agreement with the hydrograph from the beginning of each rainfall until the rain had stopped and the water level had begun to fall. The fluctuations in water quality in the samples taken by the automatic sampler during those rain events showed patterns similar to those of water samples taken by hand. There were also no problems with the water level or the EC sensor during the investigation periods, but the pH values were lower than those in the laboratory. The results showed that the system is suitable for taking water samples from mountainous streams during rain events.

Cimetidine increases survival of colorectal cancer patients with high levels of sialyl Lewis-X and sialyl Lewis-A epitope expression on tumour cells.

Cimetidine has been shown to have beneficial effects in colorectal cancer patients. In this study, a total of 64 colorectal cancer patients who received curative operation were examined for the effects of cimetidine treatment on survival and recurrence. The cimetidine group was given 800 mg day(-1) of cimetidine orally together with 200 mg day(-1) of 5-fluorouracil, while the control group received 5-fluorouracil alone. The treatment was initiated 2 weeks after the operation and terminated after 1 year. Robust beneficial effects of cimetidine were noted: the 10-year survival rate of the cimetidine group was 84.6% whereas that of control group was 49.8% (P<0.0001). According to our previous observations that cimetidine blocked the expression of E-selectin on vascular endothelium and inhibited the adhesion of cancer cells to the endothelium, we have further stratified the patients according to the expression levels of sialyl Lewis antigens X (sL(x)) and A (sL(a)). We found that cimetidine treatment was particularly effective in patients whose tumour had higher sL(x) and sL(a) antigen levels. For example, the 10-year cumulative survival rate of the cimetidine group with higher CSLEX staining, recognizing sL(x), of tumours was 95.5%, whereas that of control group was 35.1% (P=0.0001). In contrast, in the group of patients with no or low levels CSLEX staining, cimetidine did not show significant beneficial effect (the 10-year survival rate of the cimetidine group was 70.0% and that of control group was 85.7% (P=n.s.)). These results clearly indicate that cimetidine treatment dramatically improved survival in colorectal cancer patients with tumour cells expressing high levels of sL(x) and sL(a).

Runoff characteristics of major ionic species during rain events in forested watershed.

The runoff characteristics of major ionic species from a stream in a forested watershed were investigated during two rain events. The values of EC and the concentrations of alkalinity, anions and cations, except for NO3-, decreased according to the increase of discharge, and showed a sharp lower peak. On the other hand, the concentrations of NO3- and K+ indicated an opposite change. The amount of output of anions and cations was also larger than those of the input, especially in a storm event. During a storm event, the NO3- concentrations in soil water 20 cm deep taken by a tension lysimeter were not detected, even though the surface soil of 0-5 cm deep included 20 to 50 mg/kg of NO3-. The direct contribution for NO concentrations by suspended solids in water was estimated through three percentages of the stream water output. Surface runoff was also not observed. These results suggest that the prompt subsurface runoff of the direct runoff from surface layer of soil may be predominant during rainfall in the forested area, and the increase of NO3- concentrations in the stream may be caused through the process.

Runoff characteristics of nutrients in the forest streams in Hyogo Prefecture, Japan.

Nutrients and other pollutant runoffs from streams in artificial forest areas in central Hyogo Prefecture in southwest Japan have been investigated to estimate pollutant loads since 1995. The orthophosphate and ammonium nitrogen contents were usually low and constant during the investigation. When the flowrates of the streams were normal, the concentrations of suspended solids, COD(Mn), TOC and total phosphorus were very low, and did not change much. However, when stream flows were increased by rainstorms or other precipitation, higher concentrations of these parameters occurred. Otherwise, the average concentrations of nitrate nitrogen and total nitrogen were 0.26 mg/l and 0.31 mg/l, respectively, and they were often increased by precipitation events. They changed at the same time because the ratio of nitrate nitrogen per total nitrogen was high, about 80%. The fluctuation of concentrations of total phosphorus was similar to SS concentrations, which suggested that phosphorus was discharged in the types of suspended solids from forest areas. The specific loads of the nutrients and some other pollutants did not differ among the three watersheds investigated. However, the difference among them between fine days and rainy days was fairly large. It was presumed that pollutant runoff from forest areas is strongly dependent on precipitation events.

Heterogeneous expression of nonmuscle myosin heavy chain-B in mesangial cells of patients with Gitelman's syndrome.

AIMS: It has been suggested that angiotensin II (Ang II) promotes hypertrophy and hyperplasia of mesangial cells. Nonmuscle myosin heavy chain-B (NMHC-B) and alpha-smooth muscle (SM) actin are considered to be molecular markers for phenotypic change ofproliferative mesangial cells. One of the clinical characteristics in Gitelman's syndrome (GS) is the elevation of plasma Ang II. However, little is known about the relation between Ang II and phenotypic change of mesangial cells in patients with GS. In this report, we examined the expression of NMHC-B and alpha-SM actin in mesangial cells of two GS patients. MATERIALS AND METHODS: Plasma renin activity, and the concentrations of Ang II, 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha), urinary kallikrein, and 6-keto-PGF1alpha were measured. Immunohistochemical staining of NMHC-B and alpha-SM actin in mesangial cells of GS patients was also performed. RESULTS: Both cases of GS showed normal glomerular function, few histological abnormalities, and higher than normal plasma concentrations of renin and Ang II. Furthermore, one case showed a high urinary concentration of kallikrein and the expression of both NMHC-B and alpha-SM actin in mesangial cells. The other case showed a high urinary concentration of 6-keto-PGF1alpha but not kallikrein and without the expression of NMHC-B and alpha-SM actin. CONCLUSION: Not only plasma kinin-kallikrein and prostaglandins, but the renal expression of NMHC-B and alpha-SM actin may be variable in different patients with GS.

Characterization of T cell receptor beta chains of accumulating T cells in chronic ongoing myocarditis demonstrated by heterotopic cardiac transplantation in mice.

Autoimmne mechanisms have been implicated in the pathogenesis of chronic ongoing mycarditis. An earlier study of murine chronic ongoing myocarditis reported that infiltrating T cells and macrophages were prominent in the normal donor heart, in a heterotopic cardiac transplantation model. It was demonstrated that myocarditis was transferred to a normal heart transplanted into a mouse with chronic myocarditis. The present study investigated an autoimmune link to the pathogenesis of chronic ongoing myocarditis by analyzing the T cell clonalities in the model. To characterize the accumulating T cells in the donor heart, the T cell receptor beta genes (TCRBG) were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from mRNA in the donor hearts and accumulating TCRBG clonotypes were contrasted with those from recipient hearts. Inbred 3-week-old A/J mice were inoculated intraperitoneally with Coxsackievirus B3 (Nancy strain), 2 x 10(4) PFU, and housed for more than 60 days. Normal A/J mouse hearts were transplanted into the same strain of mice without myocarditis, as well as into the mice with chronic ongoing myocarditis. Both recipient and donor hearts were evaluated histologically 2 weeks after the transplantation. TCRBG were amplified by RT-PCR from mRNA of recipient and donor hearts and spleens. The specific accumulating TCRBG clonotypes were identified by their single strand conformation polymorphism. Multiple clonotypic accumulations occurred in the donor heart after cardiac transplantation. Distinct oligoclonal accumulation of TCR Vbeta1, 10, and 13 T cells was found in both recipient and donor hearts in 3 of 4 mice. Moreover, these clonotypes were not observed in spleen cells of the recipient mice. T specific cells expanding clonotypes of TCRBG are responsible for transferring myocarditis to the donor heart. An autoimmune response may, therefore, play a key role in the progression of chronic ongoing myocarditis.

Dilazep, a nucleoside transporter inhibitor, modulates cell cycle progression and DNA synthesis in rat mesangial cells in vitro.

The direct effects of the nucleoside transporter inhibitor dilazep on the cell cycle of mesangial cells have not before been investigated. The purpose of this study was to elucidate whether dilazep can inhibit the proliferation of mesangial cells and how it interferes with the cell cycle of these cells. DNA histograms were used and BrdUrd uptake rate was measured by flow cytometry. There was no significant difference in the cell numbers among the untreated group and the 10(-5) M, 10(-6) M or 10(-7) M dilazep-treated groups at 24 h of incubation. However, at 48 and 72 h, the cell numbers in the dilazep-treated groups were significantly lower compared with that of the untreated group (P < 0.005). The DNA histograms of cultured rat mesangial cells at 12, 24, and 48 h of incubation with 10(-5) M dilazep showed that the ratio of the S phase population in the dilazep-treated group decreased by 2.2% at 12 h, by 9.6% at 24 h, and by 18.9% at 48 h compared with the untreated group. The ratio of the G0/G1 phase population in the dilazep-treated group significantly increased: 6.8% at 12h (P < 0.05), 13.9% at 24 h (P < 0.001), and 76.5% at 48 h (P < 0.001) compared with the untreated group. A flow cytometric measurement of bivariate DNA/BrdUrd distribution demonstrated that the DNA synthesis rate in the S phase decreased after 6 h (P < 0.005) and 12 h (P < 0.05) of incubation compared with the untreated group. These results suggest that dilazep inhibits the proliferation of cultured rat mesangial cells by suppressing the G1/S transition by prolonging G2/M and through decreasing the DNA synthesis rate.

Mixed connective tissue disease with multiple organ damage: successful treatment with plasmapheresis.

A 24-year-old-woman with mixed connective tissue disease (MCTD) developed multiple organ failure, disseminated intravascular coagulation (DIC), metabolic acidosis, and respiratory and renal failure resulting from visceral vasospasm, so-called visceral Raynaud's phenomenon. After plasmapheresis, the condition of multiple organ failure was markedly improved. The successful treatment with plasmapheresis was dependent upon the removal of immune complexes in serum and improvement of visceral circulation. Thus plasma exchange is recommended as a possible a treatment for multiple organ damage in MCTD.

Eosinophil counts and plasma fibrinogen in patients with vasospastic angina pectoris.

Epidemiologic studies have suggested a relation between white blood cell (WBC) counts and the incidence of coronary heart disease. However, the relation between vasospastic angina pectoris (VAP) and WBC counts remains to be elucidated. To clarify the relation between differential and WBC counts in VAP, we compared the hematologic values, blood chemical values, plasma fibrinogen levels, C-reactive protein levels, and coronary risk factors in patients with spontaneous attacks of VAP (n = 39) with those in patients with stable effort angina pectoris (EAP, n = 35) and in control subjects (n = 19). Patients with VAP were further divided into mild VAP (n = 22) and severe VAP groups (n = 17). There were no differences in the coronary risk factors, body temperature, total WBC counts, and C-reactive protein levels among the control, EAP, mild VAP, and severe VAP groups, except that the high-density lipoprotein cholesterol in the EAP group was significantly lower than that in the control group (p <0.01). In contrast, the eosinophil counts were significantly higher in the severe VAP group than in the other 3 groups (p <0.01). Plasma fibrinogen levels were also significantly higher in the severe VAP group than in the other 3 groups (p <0.05). The follow-up study for differential and WBC counts in patients with VAP (n = 23) demonstrated that, after medical therapy, the eosinophil counts were significantly decreased to the some level as those in the control group (p <0.0001). Thus, the eosinophil counts and plasma fibrinogen levels could predict the severity of VAP. Furthermore, a follow-up study in patients with VAP suggests that coronary vasospasm could result in an increase in eosinophil counts.

Infection Behavior of Venturia nashicola, the Cause of Scab on Asian Pears.

ABSTRACT The infection of Japanese pear by Venturia nashicola, the cause of scab on Asian pears (Japanese pear, Pyrus pylifolia var. culta; Chinese pear, P. ussuriensis), was examined using light and electron microscopy to determine the mechanism of resistance in pears. Early stages of infection were similar on the susceptible cv. Kosui, the resistant cv. Kinchaku, and the nonhost European pear (P. communis) cv. Flemish Beauty. V. nashicola penetrated only the cuticle layer on pear leaves and formed subcuticular hyphae on all three cultivars. Hyphae were localized in the pectin layer of pear leaves and never penetrated into the cytoplasm of epidermal cells. This restriction of fungal growth suggested that pectinases released by infection hyphae or subcuticular hyphae may be important in infection. Subcuticular hyphae were modified ultrastructurally in the pectin layer of resistant pear cultivars accompanied by fungal cell death. In contrast, fungal cells appeared intact in susceptible pear cultivars, suggesting the existence of resistance mechanisms.

Repetitive coxsackievirus infection induces cardiac dilatation in post-myocarditic mice.

The relation between mycarditis and dilated cardiomyopathy (DCM) is controversial. To clarify the pathogenic mechanism of these diseases, the present study examined the effect of repetitive inoculation with coxsackievirus B3 (CVB3) in post-myocarditic mice. Inbred 3-week-old A/J mice were inoculated intraperitoneally with CVB3 (Nancy strain; 2x10(4) plaque-forming units) and reinfected in the same manner with CVB3 at 40 weeks (3W+/40W+). All mice were killed at 42 weeks old. The weight of the hearts of the 3W+/40W+ group were significantly increased compared with those of the 3W-/40W+ group, and both the heart weight/body weight and lung weight/body weight ratios of the 3W+/40W+ group were also significantly increased over those of the 3W-/40W- group, although the levels of serum neutralizing antibody titers were significantly increased in the 3W+/40W+ group over the level of the other groups. No increase in inflammatory cell infiltration or fibrosis progression was observed in the 3W+/40W+ group relative to the 3W+/40W- group, but the second inoculation resulted in a significant left ventricular dilatation and in left and right ventricular free wall thinning (3.31+/-0.20 mm vs 2.61+/-0.19 mm, p<0.05; 0.54+/-0.09 mm vs 0.72+/-0.16 mm, p<0.05, respectively). The sarcomere length was also significantly increased in the 3W+/40W+ group compared with that of the other groups, as determined by electron microscopy. Degenerative or necrotic areas in the infected hearts were not stained with anti-mouse IgG antibody, but were stained, only in 3W+/40W+ mice, with anti-mouse IgM antibody. The concentrations of TNF-alpha in the hearts of the 3W+/40W+ group were increased significantly over those of the 3W+/40W- group. Repetitive CVB3 infection produced cardiac dilatation without inflammatory cell infiltration in post- myocarditic mice. Autoimmunity mediated by the circulation of certain antibodies (eg, antibodies against the CVB3 genome or a CVB3-related protein) may be part of the pathogenic mechanism for this phenomenon. Thus, repetitive virus infection might contribute to the pathogenesis of cardiac dilatation.

Angiotensin II type 1 receptor antagonist downregulates nonmuscle myosin heavy chains in spontaneously hypertensive rat aorta.

The aim of this study was to clarify the differences between the angiotensin II type 1 (AT1) receptor antagonist and the angiotensin-converting enzyme (ACE) inhibitor on smooth muscle and nonmuscle myosin heavy chain isoforms in aortic smooth muscle cells of Wistar-Kyoto rats and spontaneously hypertensive rats. All 4 myosin heavy chain isoforms are heterogeneously expressed in the smooth muscle cells of the aortic tunica media in 20-week-old rats, and the contractile-type myosin heavy chains are highly expressed in smooth muscle cells of the aortic tunica media compared with the synthetic-type myosin heavy chains. Both the AT1 receptor antagonist and the ACE inhibitor had the same effects on hemodynamics, smooth muscle cell hypertrophy and proliferation, fibrosis, and vascular remodeling in spontaneously hypertensive rats. However, the AT1 receptor antagonist had a more potent effect on the downregulation of the synthetic-type myosin heavy chains than the ACE inhibitor in spontaneously hypertensive rat aortic tunica media. In contrast, these effects of the AT1 receptor antagonist and the ACE inhibitor on hemodynamics, morphology, fibrosis, and expression of myosin heavy chain isoforms in smooth muscle cells of the aortic tunica media were not observed in Wistar-Kyoto rats. Thus, within 6 weeks, the AT1 receptor antagonist might modulate the cellular composition of myosin heavy chain isoforms in smooth muscle cells more efficiently than the ACE inhibitor, without morphological changes in the spontaneously hypertensive rat aorta.

Fas expression and apoptosis correlate with cardiac dysfunction in patients with dilated cardiomyopathy.

Fas is a transmembranous glycoprotein that mediates apoptosis. To elucidate the roles of Fas and of myocyte apoptosis in patients with dilated cardiomyopathy (DCM), the expression of Fas and the fragmentation of DNA were compared in endomyocardial biopsy specimens obtained from patients with DCM. Endomyocardial biopsy was performed on 19 subjects (16 with DCM and 3 control subjects) who also underwent cardiac catheterization and echocardiography. Fas and bcl-2 expression were assayed immunohistochemically, and in situ TdT staining was performed to estimate the number of apoptotic cells. Samples from the DCM patients stained more intensely with anti-Fas antibody than those from control patients (p<0.05). The percentage of in situ TdT-positive cells was significantly higher in the DCM group than in the control group (p<0.05). A correlation between Fas expression and in situ TdT staining was observed in 67% of myocytes in the DCM group. Moreover, the percentage of in situ TdT staining was significantly higher in subjects with severely impaired left ventricular systolic function than in those whose systolic function was mild to moderately impaired, or who had normal systolic function (p<0.05). The samples showed little expression of bcl-2. These results suggest that Fas expression and apoptosis may be involved in the progression of cardiac dysfunction in DCM.