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An 81-year-old female was referred to our department 1 year ago due to a worsening renal function. Her manifestations met the criteria of Sjögren syndrome, suggesting renal failure likely resulting from tubulointerstitial nephritis (TIN) due to Sjögren syndrome. However, at her request, she was followed up with no further investigation or treatment. The following July, since her renal function deteriorated again, a renal biopsy was performed. Using IgM-CD138 dual staining, the renal pathology showed the infiltration of accumulated IgM-positive plasma cells within the renal insterstitium, so she was diagnosed with tubulointerstitial nephritis with IgM-positive plasma cells (IgMPC-TIN).IgMPC-TIN, proposed by Takahashi et al. in 2017, as a type of TIN, is characterized by the pathological infiltrations of IgM-positive plasma cells within the renal insterstitium and is effectively treated with corticosteroid therapy. Despite her old age, corticosteroid therapy was performed, resulting in the improvement in her renal function according to blood and urine tests and an improved pulmonary involvement, although renal dysfunction remained.Elderly patients often have multiple underlying medical conditions and take numerous medications, so differentiating renal disorders is challenging. However, a renal biopsy, even in an elderly patient, can aid in identifying the cause of renal disorders and predicting the prognosis. IgMPC-TIN is a condition in which renal function can be expected to improve if treated. It is thus important to make a diagnosis of IgMPC-TIN without overlooking and to consider proper treatment.

Cancer immunotherapy using chimeric antigen receptor–armed T (CAR T) cells have been shown to improve outcomes significantly in patients with hematological malignancies. However, cytokine release syndrome (CRS) remains a risk. CRS is characterized by the excessive activation of CAR T cells and macrophages. Signs and symptoms of CRS are usually resolved after steroid administration, but steroids abrogate the expansion and persistence of CAR T cell populations. Tocilizumab is a humanized monoclonal antibody (mAb) that attenuates CRS without significant loss of CAR T cell activity. However, interleukin-6 (IL-6)/IL-6 receptor (IL-6R) blockade alone cannot relieve CRS symptoms fully, and novel treatments are needed to prevent or cure CRS. TO-207 is an N-benzoyl-L-phenylalanine derivative that significantly inhibits inflammatory cytokine production in human monocyte and macrophage-specific manner. We investigated whether TO-207 could inhibit cytokine production without impairing CAR T cell function in a CRS-simulating co-culture system.

The cerebellar lesions of bovine spongiform encephalopathy (BSE)-infected guinea pigs were characterized as severe atrophy of the cerebellar cortex associated with the loss of granule cells, decrease in the width of the molecular layer, and intense protease-resistant prion protein (PrPSc ) accumulations that are similar to cerebellar lesions in kuru and the VV2 type of sporadic Creutzfeldt-Jakob disease. The aim of this study is to assess the relationships between the distribution and localization of PrPSc and synapses expressing neurotransmitter transporters in order to reveal the pathogenesis of the disease. We used cell-type-specific immunohistochemical makers recognizing glutamatergic and γ-aminobutylic acid (GABA)ergic terminals to identify terminals impaired with PrPSc accumulations. The distribution of PrPSc accumulations and immunoreactivity of synaptic vesicles were studied throughout the neuroanatomical pathways in cerebellar lesions. Time course study demonstrated that PrPSc accumulation showed a tendency to spread from granular layer to molecular layer. The immunoreactivity of vesicular glutamate transporter 1 (VGluT1) was localized in axon terminals of cerebellar granule cells, and decreased in association with the severity of PrPSc accumulations and loss of granule cells. Immunoreactivities of vesicular glutamate transporter 2 (VGluT2) and vesicular GABA transporter (VGAT) that exist in axon terminals of inferior olivary neurons and GABAergic synapses of Purkinje cells, respectively, were preserved well in these lesions. In brainstem, VGluT1 immunoreactivity decreased selectively in pontine nuclei that are a component of the pontocerebellar pathway, although other neurotransmitter immunoreactivities were preserved well. Our findings suggest that the selective loss of VGluT1-immunoreactive synapses subsequent to PrPSc accumulations can contribute to the pathogenesis of cerebellar lesions of BSE-infected guinea pigs.

The protozoan Cryptosporidium occurs in a wide range of animal species including many Cervidae species. Fecal samples collected from the Hokkaido sika deer (Cervus nippon yesoensis), a native deer of Hokkaido, in the central, western, and eastern areas of Hokkaido were examined by polymerase chain reaction (PCR) to detect infections with Cryptosporidium and for sequence analyses to reveal the molecular characteristics of the amplified DNA. DNA was extracted from 319 fecal samples and examined with PCR using primers for small-subunit ribosomal RNA (SSU-rRNA), actin, and 70-kDa heat shock protein (HSP70) gene loci. PCR-amplified fragments were sequenced and phylogenetic trees were created. In 319 fecal samples, 25 samples (7.8 %) were positive with SSU-rRNA PCR that were identified as the Cryptosporidium deer genotype. Among Cryptosporidium-positive samples, fawns showed higher prevalence (16.1 %) than yearlings (6.4 %) and adults (4.7 %). The result of Fisher's exact test showed a statistical significance in the prevalence of the Cryptosporidium deer genotype between fawn and other age groups. Sequence analyses with actin and HSP70 gene fragments confirmed the SSU-rRNA result, and there were no sequence diversities observed. The Cryptosporidium deer genotype appears to be the prevalent Cryptosporidium species in the wild sika deer in Hokkaido, Japan.

Emerging tick-borne diseases (TBDs) are important foci for human and animal health worldwide. However, these diseases are sometimes over looked, especially in countries with limited resources to perform molecular-based surveys. The aim of this study was to detect and characterize spotted fever group (SFG) rickettsiae and Anaplasmataceae in Bangladesh, which are important tick-borne pathogens for humans and animals worldwide. A total of 50 canine blood samples, 15 ticks collected from dogs, and 154 ticks collected from cattle were screened for the presence of SFG rickettsiae and Anaplasmataceae using molecular-based methods such as PCR and real-time PCR. The sequence analysis of the amplified products detected two different genotypes of SFG rickettsiae in ticks from cattle. The genotype detected in Rhipicephalus microplus was closely related to Rickettsia monacensis, while the genotype detected in Haemaphysalis bispinosa was closely related to Rickettsia sp. found in Korea and Japan. Anaplasma bovis was detected in canine blood and ticks (Rhipicephalus sanguineus and H. bispinosa). Unexpectedly, the partial genome sequence of Wolbachia sp., presumably associated with the nematode Dirofilaria immitis, was identified in canine blood. The present study provides the first molecular evidence of SFG rickettsiae and A. bovis in Bangladesh, indicating the possible emergence of previously unrecognized TBDs in this country.

Studies have shown that liquid crystals structurally resembling the intercellular lipids in the stratum corneum can beneficially affect the skin when applied topically by stimulating the skin’s natural regenerative functions and accelerating epidermal turnover. In the present study, the effects of applying low concentrations of a liquid crystal gel of our own creation were evaluated using epidermal thickening in mouse skin as an assay for effective stimulation of epidermal turnover. A liquid crystal gel was also applied topically to human facial skin, and analysis was conducted using before-and-after photographs of age spots, measurements of L* values that reflect degree of skin pigmentation, single-layer samples of the stratum corneum obtained via tape-stripping, and measurements of trans-epidermal water loss that reflect the status of the skin’s barrier function. The results suggested that cost-effective creams containing as low as 5% liquid crystal gel might be effective and safely sold as skin care products targeting age spots and other problems relating to uneven skin pigmentation.

Myofibroblast accumulation is a pathological feature of lung diseases requiring oxygen therapy. One possible source for myofibroblasts is through the epithelial-to-mesenchymal transition (EMT) of alveolar epithelial cells (AEC). To study the effects of oxygen on alveolar EMT, we used RLE-6TN and ex vivo lung slices and found that hyperoxia (85% O2, H85) decreased epithelial proteins, presurfactant protein B (pre-SpB), pro-SpC, and lamellar protein by 50% and increased myofibroblast proteins, α-smooth muscle actin (α-SMA), and vimentin by over 200% ( P < 0.05). In AEC freshly isolated from H85-treated rats, mRNA for pre-SpB and pro-SpC was diminished by ∼50% and α-SMA was increased by 100% ( P < 0.05). Additionally, H85 increased H2O2 content, and H2O2 (25–50 μM) activated endogenous transforming growth factor-β1 (TGF-β1), as evident by H2DCFDA immunofluorescence and ELISA ( P < 0.05). Both hyperoxia and H2O2 increased SMAD3 phosphorylation (260% of control, P < 0.05). Treating cultured cells with TGF-β1 inhibitors did not prevent H85-induced H2O2 production but did prevent H85-mediated α-SMA increases and E-cadherin downregulation. Finally, to determine the role of TGF-β1 in hyperoxia-induced EMT in vivo, we evaluated AEC from H85-treated rats and found that vimentin increased ∼10-fold ( P < 0.05) and that this effect was prevented by intraperitoneal TGF-β1 inhibitor SB-431542. Additionally, SB-431542 treatment attenuated changes in alveolar histology caused by hyperoxia. Our studies indicate that hyperoxia promotes alveolar EMT through a mechanism that is dependent on activation of TGF-β1 signaling.

Cancer immunotherapy using chimeric antigen receptor-armed T cells (CAR-T cells) have shown excellent outcomes in hematological malignancies. However, cytokine release syndrome (CRS), characterized by excessive activation of CAR-T cells and macrophages remains to be overcome. Steroid administration usually resolves signs and symptoms of CRS but abrogates CAR-T cell expansion and persistence. Tocilizumab, a humanized monoclonal antibody against interleukin-6 receptor (IL-6R), attenuates CRS without significant loss of CAR-T cell activities, while perfect rescue of CRS symptoms cannot be achieved by IL-6/IL-6R blockade. There is actual need for novel strategies to prevent or cure CRS. TO-207, an N-benzoyl-L-phenylalanine derivative compound, significantly inhibits inflammatory cytokine production in a human monocyte/macrophage-specific manner. Here we tested TO-207 for its ability to inhibit cytokine production without impaired CAR-T cell function in a CRS-simulating co-culture system consisting of CAR-T cells, target leukemic cells and monocytes. To observe a precise pattern of cytokine release from CAR-T cells and monocytes, we first established a co-culture system that mimics CRS using K562/CD19 cells, 19-28z CAR-T cells, and peripheral blood CD14+ cells. IFN-γ was produced exclusively from CAR-T cells, and TNF-α, MIP-1α, M-CSF, and IL-6 were produced from both CAR-T cells and monocytes, but monocytes were the major source of these cytokine production. MCP-1, IL-1β, IL-8, and IL-10 were released exclusively from monocytes. To observe the effect of drugs on cytokine production, prednisolone (PSL), TO-207, tocilizumab, and anakinra (an IL-1R antagonist) were added to the co-culture. PSL exhibited suppressive effects on TNF-α and MCP-1 production. Tocilizumab did not suppress these cytokines. Anakinra up-regulated IL-6 and IL-1β production, probably due to activation of negative feedback loops. Interestingly, TO-207 widely suppressed all of these monocyte-derived cytokines including TNF-α, IL-6, IL-1β, MCP-1, IL-8, and GM-CSF. Next, we observed whether the cytokine inhibition by TO-207 attenuates killing effect of CAR-T cells. PSL attenuated killing effect of CD4+ CAR-T cells and CD8+ CAR-T cells toward K562/CD19 cells. In contrast, TO-207 did not exhibit any change in cytotoxicity of CD4+ CAR-T cells and CD8+ CAR-T cells. To determine whether the effect of PSL and TO-207 on cytotoxicity changes in the presence of CD14+ monocytes, CD14+ cells were added to the co-culture. In the absence of CAR-T cells, PSL induced a modest attenuation of cytotoxicity, whereas to the CAR-T cells, PSL exhibited a significant attenuation of cytotoxicity. TO-207 exhibited a minimal effect on cytotoxicity in the absence or presence of CAR-T cells. These results suggested that CAR-T cells play a major role in the cytotoxicity toward leukemia cells, and drugs that do not affect CAR-T cell functions, such as TO-207, maintain their cytotoxic effects on leukemia cells. In conclusion, our present co-culture model with K562/CD19 cells, 19-28z CAR-T cells, and CD14+ monocytes accurately recapitulate killing effect and cytokine release profiles. IFN-γ was produced exclusively by CAR-T cells, but majority of other cytokines such as TNF-α, MIP-1α, M-CSF, IL-6, MCP-1, IL-1β, IL-8, and IL-10 were from CD14+ monocytes/macrophages. Because killing effect was largely dependent on CAR-T cells while cytokine production was dependent on monocytes/macrophages, selective inhibition of pro-inflammatory cytokines from monocytes by TO-207 would be ideal for treatment of CAR-T-related CRS. These results encourage us to consider a clinical application for CRS. Figure Disclosures Futami: Torii Pharmaceutical: Research Funding. Suzuki:Torii Pharmaceutical: Employment. Kato:Torii Pharmmaceutical: Research Funding. Tahara:Torii Pharmaceutical: Employment. Imai:Celgene: Honoraria, Research Funding; Janssen Pharmaceutical K.K: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Mimura:Torii Pharmaceutical: Employment. Watanabe:Torii Pharmaceutical: Employment. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding.

Nanoporous polyethylene (PE) membranes prepared by etching treatment for diblock copolymers composed of PE and polystyrene (PS) are applicable for biotechnological interfaces of various devices. The conventional control of pore size is limited by block compositions or etching conditions. In this study, an increase in the removable PS component was enabled by blending the above base copolymer (PE-b-PS) with PS homopolymer or another block copolymer containing a greater PS content. A series of films of these blends were isothermally crystallized under the optimum conditions, and were mildly etched to prepare nanoporous membranes. Therefore, excellent robustness was achieved in the resultant nanoporous membranes, which is beneficial for wider applications. A desirable combination of precise pore dimensions and large pore volume was achieved by blending the PS homopolymer with lower molecular weight, compared to a nanoporous membrane prepared from pure base copolymer, which is also confirmed by molecular permeation tests.

"RAKUGACKY" is an interactive media system that could generate sounds from a hand-drawn sketch. It is necessary to analyze a sketch to generate sounds suitable for the sketch. The former system used colors and simple shape feature values of objects in a sketch such a circularity and an area, to recognize each object. Thus the system could recognize only simple objects in a sketch. In this paper, we improve our system to use more complex features of objects and recognize complex objects in a sketch by extracting and learning features of complex objects in a machine learning method. For example, the improved method could analyze hand-drawn animals such as cat, dog and sheep, and could synthesize cries of each animal interactively.

Ultrahigh molecular weight polyethylene (UHMW-PE) films having different molecular weights (MWs) were melt-drawn at 150 °C. The stress-strain curve for higher-MW film exhibits higher stress on the characteristic plateau region and a subsequent steeper increase of stress due to strain hardening. Structural changes during such melt-drawing were analyzed using in situ wide-angle X-ray diffraction measurements. Hexagonal crystallization occurs at the beginning of the plateau region, independent of the sample MW. Once this hexagonal reflection intensity is saturated, it remains constant even at the later stage of draw. In contrast, orthorhombic reflection intensities gradually increase with increasing draw strain. Both of these oriented crystallizations into plateau hexagonal and increasing orthorhombic forms are accelerated with increasing MW. Correspondingly, the higher amount of extended chain crystals (ECCs) was confirmed from morphological observation for the resultant melt-drawn films of the higher-MW sample. Deep entanglements can effectively transmit the applied stress; thus, the oriented amorphous melts induce rapid hexagonal crystallization with disentangling shallow entanglements, which subsequently transforms into orthorhombic form. Such hexagonal crystallization plays the role of a thermodynamic pathway for growing such ECCs, where the stable orthorhombic form gradually accumulates with increasing draw strain.

The viability of Cryptosporidium parvum oocysts and Ascaris suum eggs inoculated into aerobic and anaerobic digesters was measured. The digesters were maintained at 37 degrees C, 47 degrees C, and 55 degrees C, with 10-day detention times. Eggs and oocysts were added to each digester in a single spike or in chambers placed in the digesters for varying periods. Oocysts were inactivated very rapidly in all systems as determined by a dye permeability assay, > 99% inactivated after 10 days at 37 degrees C, 4 days at 47 degrees C, and 2 days at 55 degrees C. Eggs were more rapidly inactivated in anaerobic digesters than in aerobic digesters. At 55 degrees C, eggs in both anaerobic and aerobic digesters were > 99% inactivated within 1 h. At 47 degrees C, anaerobic digestion inactivated around 95% eggs in 2 days, but around 25% of the eggs were still viable after 10 days in aerobic digesters. At 37 degrees C, anaerobic digestion inactivated more than 75% of the eggs after 10 days, but in the aerobic digester at 37 degrees C, 10 days of treatment had no effect on viability. The oocysts and eggs added in chambers appeared to behave similarly to these pathogens added directly to the biosolids within the digesters.

A preliminary molecular epidemiological study was carried out to investigate the utility of the Cryptosporidium oocyst wall protein (COWP) gene in the detection of Cryptosporidium oocysts in fecal samples. A nested polymerase chain reaction (PCR) approach using COWP gene primers was adopted for this purpose. Fecal samples were spiked with each of 1, 10, and 100 oocysts of C. parvum, four samples for each number, and the DNA was extracted from each sample using a glassbead method. The presence of oocysts was determined using the nested PCR with COWP gene primers, and the limit of detection of oocysts by the PCR was determined. The limit of detection was 100 oocysts spiked in 1 ml of fecal material (50% sold material) (four positives/four samples tested). Seventy-five percent of DNA extracted samples spiked with 1 and 10 oocysts was positive by the PCR (three positives/four samples tested). Based on this, small sample size using the COWP gene primers with a nested PCR analysis could reliably identify infected animals rather conveniently and accurately.

The effects of freeze-thaw events on the inactivation of Cryptosporidium parvum oocysts in soil were examined. Oocysts were inoculated into distilled water in microcentrifuge tubes or into chambers containing soil the water content of which was maintained at 3%, 43%, or 78% of the container capacity. The chambers and tubes were then embedded in 3 soil samples from different aspects of a hillside landscape (Experiments 1 and 2) and in 3 distinct soil types (Experiment 3) and frozen at -10 C. Containers were thawed every 3 days for a period of 24 hr in 1-9 freeze-thaw cycles over 27 days (Experiments 1 and 2) and 2-5 freeze-thaw cycles over 15 days (Experiment 3). Oocyst viability was measured using the fluorescent dyes 4'6-diaminidino-2-phenylindole and propidium iodide. Inactivation rates were greater in soils than in water and greater in dry soil than in moist and wet soils. Soil type showed no effect on inactivation. Oocysts subjected to freeze-thaw cycles had inactivation rates not significantly different from those of oocysts subjected to -10 C under static conditions. The results indicated that 99% of oocysts exposed to soils that are frozen at -10 C will become inactivated within 50 days whether or not freeze-thaw cycles occur.

Cryptosporidium parvum oocyst viability was determined by a dye permeability assay using a flow cytometric method. Oocysts were inoculated into small chambers with soil and biosolids. Oocysts extracted from soil and biosolids were then stained with propidium iodide (PI) and labeled with a fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody. The oocyst population in each sample was determined using forward and side scatter plots, then further analyzed with fluorescence. A red and green fluorescence detector using gates established single populations of unstained, PI-stained, or FITC-labeled oocysts. No statistical difference was observed between viability of oocysts extracted from soil and biosolids as determined by either flow cytometry or microscopy. The location of excysted oocysts was changed in forward and side scatter plots. Results indicated that, although oocysts are not identified if they excyst, the flow cytometric method could be used to determine oocyst viability from spiked environmental samples.

Cryptosporidium parvum oocysts were examined to ascertain excystation requirements and the effects of gamma irradiation. Oocysts and excysted sporozoites were examined for dye permeability and infectivity. Maximum excystation occurred when oocysts were pretreated with acid and incubated with bile salts, and potassium or sodium bicarbonate. Pretreatment with Hanks' balanced salt solution or NaCl lowered excystation; however, this effect was overcome with acid. Sodium ions were replaceable with potassium ions, and sodium bicarbonate was replaceable with sodium phosphate. Oocysts that received 200 krad irradiation excysted at the same rates as nonirradiated oocysts (95%), the excystation rates were lowered (50%) by 2,000 krad, and no excystation was observed by 5,000 krad. No differences were observed between the propidium iodide (PI) permeability of untreated oocysts and oocysts treated with 200 krad, while 92% of oocysts were PI positive after 2,000 krad. Most of the sporozoites exposed to 2,000 krad were not viable as indicated by the dye permeability assay. The oocysts irradiated with 200 and 2,000 krad infected cells, but no replication was observed. The results suggest that gamma-irradiated oocysts may still be capable of excystation and apparent infection; however, because the sporozoites could not reproduce they must not have been viable. The identification of the mechanisms that trigger and carry out the process of excystation is critical for understanding how sporozoites are freed from oocysts prior to initiating the pene- tration of the hosts' intestinal cells. In vitro excystation is a parameter that has been used to measure the viability of spo- rozoites within oocysts (Campbell et al., 1992; Jenkins et al., 1997). Jenkins et al. (1997) reported that the in vitro excystation assay for potential infectivity of oocysts correlated with the in vivo mouse infectivity test and the in vitro dye permeability assay. Several studies have examined the requirements for Crypto- sporidium parvum oocyst excystation (Fayer and Leek, 1984

This study examined the potential efficacy of Chenopodium ambrosioides as a vermifuge in lambs, testing both the essential oil and dried plant tissue. Oral administration of the oil produced no toxic effects of importance, although the active ingredient, ascaridole, was observed in the blood. A significant reduction in the number of Trichostrongyle eggs per gram of feces was observed in treated lambs as compared with control lambs, but treated lambs did continue to shed eggs in feces. Feed intake of lambs fed dried plants was significantly less than that of control lambs apparently due to an aversion to the odor of the plants and the unusual nature of the plant material as presented.

COP1 is a Ring-Finger E3 ubiquitin ligase that is involved in plant development, mammalian cell survival, growth, and metabolism. Here we report that COP1, whose expression is enhanced by insulin, regulates FoxO1 protein stability. We found that in Fao hepatoma cells, ectopic expression of COP1 decreased, whereas knockdown of COP1 expression increased the level of endogenous FoxO1 protein without impacting other factors such as C/EBPα and CREB (cAMP-response element-binding protein). We further showed that COP1 binds FoxO1, enhances its ubiquitination, and promotes its degradation via the ubiquitin-proteasome pathway. To determine the biological significance of COP1-mediated FoxO1 protein degradation, we have examined the impact of COP1 on FoxO1-mediated gene expression and found that COP1 suppressed FoxO1 reporter gene as well as FoxO1 target genes such as glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two key targets for FoxO1 in the regulation of gluconeogenesis, with corresponding changes of hepatic glucose production in Fao cells. We suggest that by functioning as a FoxO1 E3 ligase, COP1 may play a role in the regulation of hepatic glucose metabolism.

TRB3 is a pseudokinase whose expression is regulated during stress response and changing of nutrient status. TRB3 negatively regulates Akt activation and noticeably, TRB3 expression is induced by insulin. Here, we sought to determine the dynamic relationship between TRB3 expression and Akt activation. We find that insulin induces TRB3 expression in cell type dependent manner such that in hepatic cells and adipocytes but not Beta cells and muscle cells. In Fao hepatoma cells, induction of TRB3 expression by insulin restrains Akt activation and renders Akt refractory to further activation. In addition, we have also analyzed the roles of PI3K and its downstream kinases Akt and atypical PKC in TRB3 expression. Induction of TRB3 expression by insulin requires PI3K. However, inactivation of Akt enhances TRB3 expression whereas inhibition of PKCzeta expression impairs TRB3 expression induced by insulin. Our data demonstrated that PI3K conveys both negative and positive signals to TRB3 expression. We suggest that insulin-induced TRB3 expression functions as an indicator how multiple insulin-induced signal transduction pathways are balanced.

Members of Akt family are highly conserved protein kinase and yet, they show clearly distinct in vivo functions. Here, we have examined the abilities of Akt1 and Akt2 to activate CREB. We found that, in contrast to Akt1 that induces CREB phosphorylation at Ser-133 and CREB target gene expression, Akt2 was unable to induce CREB phosphorylation at Ser-133 in vivo and CREB target gene expression. This difference is specific to CREB as both Akt1 and Akt2 similarly inhibits FoxO1 mediated gene expression. We further showed that the regulatory domain of Akt plays a critical role to confer Akt substrate specificity as substitution of regulatory domain of Akt1 with that of Akt2 abolished the ability of Akt1 to activate CREB. We suggest that the regulatory domain of Akts contributes to the functional difference between Akt1 and Akt2.

TRB3 is a member of TRB protein family characterized by containing a variant kinase domain without enzymatic activity. Interacting with Ser/Thr protein kinases Akt, TRB3 impairs Akt activation induced by growth factors and insulin. In this study we have examined the potential role of TRB3 in muscle differentiation. Our data indicated that the expression of TRB3 is downregulated during C2C12 cells undergoing muscle differentiation and that overexpression of TRB3 inhibits Akt activation during differentiation. Correspondingly, overexpression of TRB3 inhibits, while knockdown TRB3 enhances C2C12 differentiation. Thus, our studies indicated that TRB3 plays a critical role in muscle differentiation.

The need exists to understand the environmental parameters that affect inactivation of Cryptosporidium parvum oocysts in soil under field conditions. The inactivation of C. parvum oocysts placed in the natural environment was studied at a dairy farm in western New York State, USA. Seventy sampling points were arranged in a grid with points 150 m apart using the Geographic Information System. The sampling points were distributed among three distinct areas: woodland, corn field and pasture. Purified oocysts were inoculated into chambers filled with soil from each sampling point, and buried in the surface of each respective sampling point. To compare C. parvum oocyst survival with another organism known to survive environmental stresses, Ascaris suum eggs were also placed in soil contained in chambers and buried at the same sampling points as the oocysts. As controls oocysts and eggs in distilled water were also placed at each sampling point. Oocyst and egg viability, soil pH and percent gravimetric water content were measured at all sampling points at 0, 60 and 120 day sampling periods. Soil organic content was determined for each sampling point. At 120 days after placement, mean viability of C. parvum oocysts was 10% although at a few sampling points, 30% of oocysts were still potentially infective; whereas 90% of A. suum eggs were viable at all sampling points. Statistically significant differences were not observed among the three different sampling areas, and no statistically significant predictors were found by regression analysis. Results exemplified the heterogeneity of soil parameters and oocyst viability across a landscape; such results make predictive models for C. parvum inactivation problematical. The long-term survival of C. parvum oocysts in soil under field conditions, as this study demonstrated, emphasizes their potential as a risk to contaminate surface waters.