High-Speed Diagnosis of Bacterial Pathogens at the Single Cell Level by Raman Microspectroscopy with Machine Learning Filters and Denoising Autoencoders.

Accurate and rapid identification of infectious bacteria is important in medicine. Raman microspectroscopy holds great promise in performing label-free identification at the single-cell level. However, due to the naturally weak Raman signal, it is a challenge to build extensive databases and achieve both accurate and fast identification. Here, we used signal-to-noise ratio (SNR) as a standard indicator for Raman data quality and performed bacterial identification using 11, 141 single-cell Raman spectra from nine bacterial strains. Subsequently, using two machine learning methods, a simple filter, and a neural network-based denoising autoencoder (DAE), we demonstrated 92% (simple filter using 1 s/cell spectra) and 84% (DAE using 0.1 s/cell spectra) identification accuracy. Our machine learning-aided Raman analysis paves the way for high-speed Raman microspectroscopic clinical diagnostics.

RCE­4, a potential anti­cervical cancer drug isolated from Reineckia carnea, induces autophagy via the dual blockade of PI3K and ERK pathways in cervical cancer CaSki cells.

The steroidal saponin RCE­4 (1ß, 3ß, 5ß, 25S)­spirostan­1, 3­diol 1­[α­L­rhamnopyranosyl­(1→2)­ß­D­xylopyranoside], isolated from Reineckia carnea, exerts significant anti­cervical cancer activity by inducing apoptosis. The potential effect of RCE­4 on proliferation inhibition and autophagy induction has rarely been studied. Therefore, the focus of the present study was to investigate the effects of RCE­4 on proliferation, and to elucidate the detailed mechanisms involved in autophagy induction in cervical cancer cells. CaSki cells were treated with RCE­4 or/and autophagy inhibitors, and the effect of RCE­4 on cellular proliferation was assessed by MTT assay. The pro­autophagic properties of RCE­4 were subsequently confirmed using monomeric red fluorescent protein­green fluorescent protein­microtubule­associated proteins 1A/1B light chain 3B (LC3) adenoviruses and CYTO­ID autophagy assays, and by assessing the accumulation of lipid­modified LC3 (LC3II). The mechanisms of RCE­4­induced autophagy were investigated by western blot analysis. The results demonstrated that inhibiting autophagy significantly promoted RCE­4­induced cell death, indicating that autophagy served a protective role following RCE­4 treatment. In addition, RCE­4­induced autophagy was reflected by increased expression levels of the serine/threonine­protein kinase ULK1, phosphorylated (p)­ULK1, p­Beclin­1 and LC3II, the formation of autophagosomes and autolysosomes, and sequestosome 1 (p62) degradation. Subsequent analysis indicated that RCE­4 activated the AMP­activated protein kinase (AMPK) pathway by upregulating AMPK and p­AMPK, and also inhibited the PI3K and extracellular signal­regulated kinase (ERK) signaling pathways by downregulating p­PI3K, p­Akt, p­mTOR, Ras, c­Raf, p­c­Raf, dual specificity mitogen­activated protein kinase kinase (MEK)1/2, p­MEK1/2 and p­Erk1/2. Additionally, with increased treatment times RCE­4 may impair lysosomal cathepsin activity and inhibit autophagy flux by suppressing the expression of AMPK, p­AMPK, ULK1, p­ULK1 and p­Beclin­1, and upregulating that of p62. These results indicated that the dual RCE­4­induced inhibition of the PI3K and ERK pathways may result in a more significant anti­tumor effect and prevent chemoresistance, compared with the inhibition of either single pathway; furthermore, dual blockade of PI3K and ERK, and the AMPK pathway may be involved in the regulation of autophagy caused by RCE­4. Taken together, RCE­4 induced autophagy to protect cancer cells against apoptosis, but AMPK­mediated autophagy was inhibited in the later stages of RCE­4 treatment. In addition, autophagy inhibition improved the therapeutic effect of RCE­4. These data highlight RCE­4 as a potential candidate for cervical cancer treatment.

FLT3-ITD drives Ara-C resistance in leukemic cells via the induction of RUNX3.

Internal tandem duplication (ITD) mutations of the FLT3 gene (FLT3-ITD) are well known to correlate with a poor prognosis in acute myeloid leukemia (AML). We previously reported that FLT3-ITD confers resistance to cytosine arabinoside (Ara-C), a key cytotoxic agent in AML treatments. In order to elucidate the detailed molecular mechanisms underlying the Ara-C resistance induced by FLT3-ITD, we performed a microarray gene expression analysis of the human leukemic cell line K562 transduced with FLT3-ITD (K562/FLT3-ITD) and identified RUNX3 as a downstream target of FLT3-ITD. The transcriptional induction of the RUNX3 expression by FLT3-ITD was noted on a Luciferase assay. The knockdown of the RUNX3 expression in the K562/FLT3-ITD cells increased the sensitivity to Ara-C, and the exogenous expression of RUNX3 per se resulted in the enhancement of Ara-C resistance in the K562 cells. A relationship between the FLT3-ITD-induced RUNX3 expression and Ara-C resistance was also observed in AML cells with an endogenous FLT3-ITD expression. Collectively, these findings demonstrate that RUNX3 is a prerequisite for Ara-C resistance via FLT3-ITD signaling.

Severe pneumonia caused by a novel influenza A (H1N1) virus in an asymptomatic emphysematous smoker.

A 49-year-old female presented with diarrhea and a high fever followed by progressive dyspnea. Until this presentation, she had been healthy except for chronic dyspepsia and diarrhea. She had a smoking habit of 15 pack-years. Laboratory tests revealed lymphopenia, hypoalbuminemia and hypogammaglobulinemia. A rapid influenza test in combination with an RT-PCR assay revealed the presence of the novel influenza A (H1N1) virus. Chest computed tomography revealed centrilobular emphysema. This report suggests that regular smoking may become a risk for severe pneumonia in patients presenting with the novel influenza A (H1N1) virus, when accompanying asymptomatic emphysema is combined with other problems such as hypoalbuminemia and hypogammaglobulinemia.

The tortoiseshell pattern in one or both sides of the submandibular glands in mucosa-associated lymphoid tissue lymphoma is related to chromosomal aberrations and the disease extent.

OBJECTIVE: Lesions of mucosa-associated lymphoid tissue (MALT) lymphoma in the submandibular glands are localized or a part of systemic involvement in association with chromosomal aberrations. This series was undertaken to investigate the sonographic features of MALT lymphoma in the submandibular glands and their relationships with chromosomal aberrations and the disease extent. METHODS: A total of 5 patients with MALT lymphoma without Sjögren syndrome in the submandibular glands were enrolled in this series. Patients underwent sonography of the submandibular glands with a high-resolution transducer before surgical biopsy of the main lesion. Sonographic characteristics of the lesions were described for their location, presence of a posterior echo, texture, and presence of an internal echo. RESULTS: Sonography in all cases showed hypoechoic and solid masses with increased posterior echo enhancement. There was an arrangement of hypoechoic small compartments demarcated by hyperechoic contour lines, which had a tortoiseshell pattern. This pattern was classified into 2 types according to its location: a lesion in the right or left side and lesions in both sides of the submandibular glands, found in 3 and 2 patients, respectively. The latter 2 cases had chromosomal aberrations of t(11;18)(q23;q23) and t(12;18)(q22;q21), respectively, and were revealed as secondary organ involvement. CONCLUSIONS: The sonographic appearance of MALT lymphoma in the submandibular glands was characterized by the tortoiseshell pattern in both primary and secondary lesions. Detection of this pattern in both sides of the submandibular glands can be an indicator of chromosomal aberrations and systematic involvement of the disease.

FLT3-ITD induces ara-C resistance in myeloid leukemic cells through the repression of the ENT1 expression.

Fms-related tyrosine kinase 3-internal tandem duplications (FLT3-ITD) are strongly associated with the refractory nature of acute myeloid leukemia (AML) by the standard combined chemotherapy. FLT3-ITD-expressing murine and human myeloid cell lines, HF6/FLT3-ITD and K562/FLT3-ITD cells, respectively, were developed in order to clarify whether FLT3-ITD is involved in the resistance to cytotoxic agents in AML. Both of these cell lines were specifically resistant to the pyrimidine analogue cytosine arabinoside (ara-C), an essential agent for AML, accompanied by the downregulation of equilibrative nucleoside transporter 1 (ENT1), a transporter responsible for the cellular uptake of ara-C. The ENT1 promoter activity and the cellular uptake of ara-C were reduced in K562/FLT3-ITD cells, and rescued by pretreating the cells with PKC412, a FLT3 inhibitor. In addition, the expression of hypoxia inducible factor 1 alpha subunit (HIF1A) transcripts was upregulated in K562/FLT3-ITD cells, and the induction of HIF-1alpha reduced the promoter activity of ENT1 gene in K562 cells. Taken together, these findings suggest that FLT3-ITD specifically induces ara-C resistance in leukemic cells by the repression of ENT1 expression, possibly through the upregulation of HIF-1alpha, while also partially accounting for the poor prognosis of AML with FLT3-ITD due to resistance to the standard chemotherapy protocols which include ara-C.

Non-caseous granulation lymphadenitis in the neck as an initial manifestation of prostate carcinoma.

We encountered a 54-year-old Japanese man who presented with painless swellings on his bilateral neck. Although ultrasonographic findings suggested metastatic lymphadenopathy, histological examination of the mass revealed non-caseous granulation lymphadenitis. He was subsequently diagnosed as having prostate carcinoma with metastasis to the multiple bones. The present case suggested that prostate carcinoma could cause cervical lymphadenopathy due to non-caseous granulation as an initial manifestation, and could be a sign for an impending clinical expression of metastasis to an area draining to the lymph nodes.