RAISING is a high-performance method for identifying random transgene integration sites.

Both natural viral infections and therapeutic interventions using viral vectors pose significant risks of malignant transformation. Monitoring for clonal expansion of infected cells is important for detecting cancer. Here we developed a novel method of tracking clonality via the detection of transgene integration sites. RAISING (Rapid Amplification of Integration Sites without Interference by Genomic DNA contamination) is a sensitive, inexpensive alternative to established methods. Its compatibility with Sanger sequencing combined with our CLOVA (Clonality Value) software is critical for those without access to expensive high throughput sequencing. We analyzed samples from 688 individuals infected with the retrovirus HTLV-1, which causes adult T-cell leukemia/lymphoma (ATL) to model our method. We defined a clonality value identifying ATL patients with 100% sensitivity and 94.8% specificity, and our longitudinal analysis also demonstrates the usefulness of ATL risk assessment. Future studies will confirm the broad applicability of our technology, especially in the emerging gene therapy sector.

Staged treatment protocol for gait with hybrid assistive limb in the acute phase of patients with stroke.

Hybrid Assistive Limb (HAL) is a wearable human assistant cyborg-type robot that helps lower-leg movement based on bioelectrical signals detected from the voluntary movement of the person wearing it. In this study, we developed a novel staged HAL treatment protocol for patients with acute stroke. The Regain Program for Gait with HAL (RPG-HAL) was formulated in four steps, based on the severity of limb paralysis. Twenty-one patients with acute stroke received a combination treatment of RPG-HAL and conventional rehabilitation. The feasibility and safety of RPG-HAL were evaluated based on changes in physical function and activities of daily living (ADL). RPG-HAL yielded improvement in gait speed, cadence, step length, and functional ambulation category (FAC). The effect size was >0.8 in all measurements. FAC (1.90) and Barthel Index (BI) (1.92) exhibited the highest scores. Twelve out of 14 patients with FAC 0 before RPG-HAL reached the upper FAC. Thus, earlier intervention using RPG-HAL as improving physical function, ADL, and gait ability in patients with stroke.

A high-throughput detection method for the clonality of Human T-cell leukemia virus type-1-infected cells in vivo.

Approximately 10-20 million of Human T-cell leukemia virus type-1 (HTLV-1)-infected carriers have been previously reported, and approximately 5% of these carriers develop adult T-cell leukemia/lymphoma (ATL) with a characteristic poor prognosis. In Japan, Southern blotting has long been routinely performed for detection of clonally expanded ATL cells in vivo, and as a confirmatory diagnostic test for ATL. However, alternative methods to Southern blotting, such as sensitive, quantitative, and rapid analytical methods, are currently required in clinical practice. In this study, we developed a high-throughput method called rapid amplification of integration site (RAIS) that could amplify HTLV-1-integrated fragments within 4 h and detect the integration sites in > 0.16% of infected cells. Furthermore, we established a novel quantification method for HTLV-1 clonality using Sanger sequencing with RAIS products, and the validity of the quantification method was confirmed by comparing it with next-generation sequencing in terms of the clonality. Thus, we believe that RAIS has a high potential for use as an alternative routine molecular confirmatory test for the clonality analysis of HTLV-1-infected cells.

In Vitro Activity of Lascufloxacin against Streptococcus pneumoniae with Mutations in the Quinolone Resistance-Determining Regions.

Lascufloxacin showed potent activity against Streptococcus pneumoniae with a GyrA or ParC mutation (first-step mutant). The frequency of selecting resistant strains tended to be lower for lascufloxacin than for levofloxacin and garenoxacin after drug exposure in first-step mutants but was similar in the comparison between lascufloxacin and moxifloxacin. The increase in MIC was smaller for lascufloxacin than for levofloxacin, garenoxacin, and moxifloxacin when clinical strains with only ParC mutations were exposed to the corresponding drug.

The Hybrid Assistive Limb® intervention for a postoperative patient with spinal dural arteriovenous fistula and chronic spinal cord injury: A case study.

CONTEXT: The purpose of this report was to describe the improvement in walking ability using the Hybrid Assistive Limb® (HAL®) intervention in the case of a patient with paraplegia after spinal cord injury whose condition deteriorated because of a spinal dural arteriovenous fistula (SDAVF). FINDINGS: A 48-year-old man started the HAL® intervention twice per week (total 10 sessions), after his neurologic improvement had plateaued from 3 to 6 months postoperatively for an SDAVF. During the HAL® intervention, the 10-m walk test (10MWT) without HAL® was performed before and after each session. An electromyography system was used to evaluate muscle activity of both the gluteus maximus (Gmax) and quadriceps femoris (Quad) muscles in synchronization with the Vicon motion capture system. The International Standards for Neurological and Functional Classification of Spinal Cord Injury (ISNCSCI) motor scores of the lower extremities and the Walking Index for Spinal Cord Injury II (WISCI II) score were also assessed to evaluate motor function. The HAL® intervention improved gait speed and cadence during the 10MWT. Before the intervention, both the Gmax and left Quad muscles were not activated. After the intervention, the right Gmax and both Quad muscles were activated in stance phase rhythmically according to the gait cycle. The ISNCSCI motor score also improved from 14 to 16, and the WISCI II scored improved from 7 to 12. CONCLUSION/CLINICAL RELEVANCE: Our experience with this patient suggests that the HAL® can be an effective tool for improving functional ambulation in patients with chronic spinal cord injury.

Unexpected effects of azole transporter inhibitors on antifungal susceptibility in Candida glabrata and other pathogenic Candida species.

The pathogenic fungus Candida glabrata is often resistant to azole antifungal agents. Drug efflux through azole transporters, such as Cdr1 and Cdr2, is a key mechanism of azole resistance and these genes are under the control of the transcription factor Pdr1. Recently, the monoamine oxidase A (MAO-A) inhibitor clorgyline was shown to inhibit the azole efflux pumps, leading to increased azole susceptibility in C. glabrata. In the present study, we have evaluated the effects of clorgyline on susceptibility of C. glabrata to not only azoles, but also to micafungin and amphotericin B, using wild-type and several mutant strains. The addition of clorgyline to the culture media increased fluconazole susceptibility of a C. glabrata wild-type strain, whereas micafungin and amphotericin B susceptibilities were markedly decreased. These phenomena were also observed in other medically important Candida species, including Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida krusei. Expression levels of CDR1, CDR2 and PDR1 mRNAs and an amount of Cdr1 protein in the C. glabrata wild-type strain were highly increased in response to the treatment with clorgyline. However, loss of Cdr1, Cdr2, Pdr1, and a putative clorgyline target (Fms1), which is an ortholog of human MAO-A, or overexpression of CDR1 did not affect the decreased susceptibility to micafungin and amphotericin B in the presence of clorgyline. The presence of other azole efflux pump inhibitors including milbemycin A4 oxime and carbonyl cyanide 3-chlorophenylhydrazone also decreased micafungin susceptibility in C. glabrata wild-type, Δcdr1, Δcdr2, and Δpdr1 strains. These findings suggest that azole efflux pump inhibitors increase azole susceptibility but concurrently induce decreased susceptibility to other classes of antifungals independent of azole transporter functions.

Roles of vacuolar H+-ATPase in the oxidative stress response of Candida glabrata.

Vacuolar H(+)-ATPase (V-ATPase) is responsible for the acidification of eukaryotic intracellular compartments and plays an important role in oxidative stress response (OSR), but its molecular bases are largely unknown. Here, we investigated how V-ATPase is involved in the OSR by using a strain lacking VPH2, which encodes an assembly factor of V-ATPase, in the pathogenic fungus Candida glabrata The loss of Vph2 resulted in increased H2O2 sensitivity and intracellular reactive oxygen species (ROS) level independently of mitochondrial functions. The Δvph2 mutant also displayed growth defects under alkaline conditions accompanied by the accumulation of intracellular ROS and these phenotypes were recovered in the presence of the ROS scavenger N-acetyl-l-cysteine. Both expression and activity levels of mitochondrial manganese superoxide dismutase (Sod2) and catalase (Cta1) were decreased in the Δvph2 mutant. Phenotypic analyses of strains lacking and overexpressing these genes revealed that Sod2 and Cta1 play a predominant role in endogenous and exogenous OSR, respectively. Furthermore, supplementation of copper and iron restored the expression of SOD2 specifically in the Δvph2 mutant, suggesting that the homeostasis of intracellular cupper and iron levels maintained by V-ATPase was important for the Sod2-mediated OSR. This report demonstrates novel roles of V-ATPase in the OSR in C. glabrata.

Contribution of the Slt2-regulated transcription factors to echinocandin tolerance in Candida glabrata.

Echinocandin-class antifungals, including micafungin, are considered as the first-line treatment for Candida glabrata infections. However, recent epidemiological surveys have revealed an increasing number of C. glabrata isolates exhibiting decreased echinocandin susceptibilities. The Slt2 mitogen-activated protein kinase pathway is important for maintenance of cell wall integrity in fungi. Rlm1 and Swi4-Swi6 cell cycle box binding factor (SBF) are transcription factors downstream of Slt2. While Slt2 and Rlm1 play important roles in response to cell wall stresses, such as micafungin exposure, little is known about SBF in C. glabrata. Here, we generated C. glabrata strains lacking or overexpressing SWI4 and SWI6 and evaluated their susceptibilities to micafungin. Micafungin tolerance considerably decreased in the ∆swi4 strain, whereas it increased in the strains overexpressing SWI4. On the other hand, deletion of SWI6 slightly impaired micafungin tolerance, but overexpression of SWI6 had no effect. These results suggest that, although Swi4 and Swi6 form a protein complex, Swi4 is involved in micafungin tolerance more predominantly than Swi6 in C. glabrata. Furthermore, the overexpression of RLM1 induced increased micafungin tolerance in the wild-type background but not in the ∆swi4 and ∆swi6 strains, suggesting that Rlm1 and SBF function interdependently in response to micafungin exposure.

The heme-binding protein Dap1 links iron homeostasis to azole resistance via the P450 protein Erg11 in Candida glabrata.

The pathogenic fungus Candida glabrata is relatively resistant to azole antifungals, which target lanosterol 14α-demethylase (Erg11p) in the ergosterol biosynthesis pathway. Our study revealed that C. glabrata exhibits increased azole susceptibility under low-iron conditions. To investigate the molecular basis of this phenomenon, we generated a strain lacking the heme (iron protoporphyrin IX)-binding protein Dap1 in C. glabrata. The Δdap1 mutant displayed growth defects under iron-limited conditions, decreased azole tolerance, decreased production of ergosterol, and increased accumulation of 14α-methylated sterols lanosterol and squalene. All the Δdap1 phenotypes were complemented by wild-type DAP1, but not by DAP1(D91G) , in which a heme-binding site is mutated. Furthermore, azole tolerance of the Δdap1 mutant was rescued by exogenous ergosterol but not by iron supplementation alone. These results suggest that heme binding by Dap1 is crucial for Erg11 activity and ergosterol biosynthesis, thereby being required for azole tolerance. A Dap1-GFP fusion protein predominantly localized to vacuolar membranes and endosomes, and the Δdap1 cells exhibited aberrant vacuole morphologies, suggesting that Dap1 is also involved in the regulation of vacuole structures that could be important for iron storage. Our study demonstrates that Dap1 mediates a functional link between iron homeostasis and azole resistance in C. glabrata.

Functional characterization of the regulators of calcineurin in Candida glabrata.

The serine-threonine-specific protein phosphatase calcineurin is a key mediator of various stress responses in fungi. Herein, we characterized functions of the endogenous regulators of calcineurin (RCNs), Rcn1 and Rcn2, in the pathogenic fungus Candida glabrata. Rcn1 exerted both inhibitory and stimulatory effects on calcineurin signaling, but Rcn2 displayed only inhibitory activity. Phenotypic analyses of C. glabrata strains lacking either RCNs, calcineurin, or both revealed that calcineurin requires Rcn1, but not Rcn2, for antifungal tolerance in C. glabrata.

The glycosylphosphatidylinositol-linked aspartyl protease Yps1 is transcriptionally regulated by the calcineurin-Crz1 and Slt2 MAPK pathways in Candida glabrata.

In the pathogenic fungus Candida glabrata, the YPS1 gene, which encodes a glycosylphosphatidylinositol-linked aspartyl protease, is required for cell wall integrity and virulence. Although the expression of YPS1 has been studied in Saccharomyces cerevisiae, the transcriptional regulation of this gene in C. glabrata is not well understood. Here, we report that C. glabrata Yps1 is required for cell growth at elevated temperatures, and that the heat-induced expression of YPS1 is regulated predominantly by the calcineurin-Crz1 pathway and partially by the Slt2 MAPK pathway. Although a total of 11 YPS genes are present in the C. glabrata genome, the loss of transcriptional induction in a calcineurin mutant was observed only for YPS1. The results of a YPS1 promoter-lacZ reporter assay using a series of constructs with mutated promoter elements indicated that the transcription factor Crz1 binds to multiple sites in the promoter region of YPS1. To date, as none of the putative Crz1 targets in C. glabrata have been characterized using a Δcrz1 mutant, monitoring the expression of YPS1 represents an effective method for measuring the activity of the calcineurin-Crz1 signaling pathway in this fungus.

Role of the Slt2 mitogen-activated protein kinase pathway in cell wall integrity and virulence in Candida glabrata.

The Slt2 mitogen-activated protein kinase pathway plays a major role in maintaining fungal cell wall integrity. In this study, we investigated the effects of SLT2 deletion and overexpression on drug susceptibility and virulence in the opportunistic fungal pathogen Candida glabrata. While the Deltaslt2 strain showed decreased tolerance to elevated temperature and cell wall-damaging agents, the SLT2-overexpressing strain exhibited increased tolerance to these stresses. A mutant lacking Rlm1, a transcription factor downstream of Slt2, displayed a cell wall-associated phenotype intermediate to that of the Deltaslt2 strain. When RLM1 was overexpressed, micafungin tolerance was increased in the wild-type strain and partial restoration of the drug tolerance was observed in the Deltaslt2 background. It was also demonstrated that echinocandin-class antifungals were more effective against C. glabrata under acidic conditions or when used concurrently with the chitin synthesis inhibitor nikkomycin Z. Finally, in a mouse model of disseminated candidiasis, the deletion and overexpression of C. glabrata SLT2 resulted in mild decreases and increases, respectively, in the CFUs from murine organs compared with the wild-type strain. These fundamental data will help in further understanding the mechanisms of cell wall stress response in C. glabrata and developing more effective treatments using echinocandin antifungals in clinical settings.

Roles of calcineurin and Crz1 in antifungal susceptibility and virulence of Candida glabrata.

A Candida glabrata calcineurin mutant exhibited increased susceptibility to both azole antifungal and cell wall-damaging agents and was also attenuated in virulence. Although a mutant lacking the downstream transcription factor Crz1 displayed a cell wall-associated phenotype intermediate to that of the calcineurin mutant and was modestly attenuated in virulence, it did not show increased azole susceptibility. These results suggest that calcineurin regulates both Crz1-dependent and -independent pathways depending on the type of stress.