Single-cell spatial landscapes of the lung tumour immune microenvironment.

Single-cell technologies have revealed the complexity of the tumour immune microenvironment with unparalleled resolution1-9. Most clinical strategies rely on histopathological stratification of tumour subtypes, yet the spatial context of single-cell phenotypes within these stratified subgroups is poorly understood. Here we apply imaging mass cytometry to characterize the tumour and immunological landscape of samples from 416 patients with lung adenocarcinoma across five histological patterns. We resolve more than 1.6 million cells, enabling spatial analysis of immune lineages and activation states with distinct clinical correlates, including survival. Using deep learning, we can predict with high accuracy those patients who will progress after surgery using a single 1-mm2 tumour core, which could be informative for clinical management following surgical resection. Our dataset represents a valuable resource for the non-small cell lung cancer research community and exemplifies the utility of spatial resolution within single-cell analyses. This study also highlights how artificial intelligence can improve our understanding of microenvironmental features that underlie cancer progression and may influence future clinical practice.

Single-cell spatial immune landscapes of primary and metastatic brain tumours.

Single-cell technologies have enabled the characterization of the tumour microenvironment at unprecedented depth and have revealed vast cellular diversity among tumour cells and their niche. Anti-tumour immunity relies on cell-cell relationships within the tumour microenvironment1,2, yet many single-cell studies lack spatial context and rely on dissociated tissues3. Here we applied imaging mass cytometry to characterize the immunological landscape of 139 high-grade glioma and 46 brain metastasis tumours from patients. Single-cell analysis of more than 1.1 million cells across 389 high-dimensional histopathology images enabled the spatial resolution of immune lineages and activation states, revealing differences in immune landscapes between primary tumours and brain metastases from diverse solid cancers. These analyses revealed cellular neighbourhoods associated with survival in patients with glioblastoma, which we leveraged to identify a unique population of myeloperoxidase (MPO)-positive macrophages associated with long-term survival. Our findings provide insight into the biology of primary and metastatic brain tumours, reinforcing the value of integrating spatial resolution to single-cell datasets to dissect the microenvironmental contexture of cancer.

A Single-Cell Atlas of Small Airway Disease in Chronic Obstructive Pulmonary Disease: A Cross-Sectional Study.

Rationale: Emerging data demonstrate that the smallest conducting airways, terminal bronchioles, are the early site of tissue destruction in chronic obstructive pulmonary disease (COPD) and are reduced by as much as 41% by the time someone is diagnosed with mild (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 1) COPD. Objectives: To develop a single-cell atlas that describes the structural, cellular, and extracellular matrix alterations underlying terminal bronchiole loss in COPD. Methods: This cross-sectional study of 262 lung samples derived from 34 ex-smokers with normal lung function (n = 10) or GOLD stage 1 (n = 10), stage 2 (n = 8), or stage 4 (n = 6) COPD was performed to assess the morphology, extracellular matrix, single-cell atlas, and genes associated with terminal bronchiole reduction using stereology, micro-computed tomography, nonlinear optical microscopy, imaging mass spectrometry, and transcriptomics. Measurements and Main Results: The lumen area of terminal bronchioles progressively narrows with COPD severity as a result of the loss of elastin fibers within alveolar attachments, which was observed before microscopic emphysematous tissue destruction in GOLD stage 1 and 2 COPD. The single-cell atlas of terminal bronchioles in COPD demonstrated M1-like macrophages and neutrophils located within alveolar attachments and associated with the pathobiology of elastin fiber loss, whereas adaptive immune cells (naive, CD4, and CD8 T cells, and B cells) are associated with terminal bronchiole wall remodeling. Terminal bronchiole pathology was associated with the upregulation of genes involved in innate and adaptive immune responses, the interferon response, and the degranulation of neutrophils. Conclusions: This comprehensive single-cell atlas highlights terminal bronchiole alveolar attachments as the initial site of tissue destruction in centrilobular emphysema and an attractive target for disease modification.

Design of an achromatic zoom metalens doublet in the visible.

Zoom metalens doublets, featuring ultra-compactness, strong zoom capability, and CMOS compatibility, exhibit unprecedented advantages over the traditional refractive zoom lens. However, the huge chromatic aberration narrows the working bandwidth, which limits their potential applications in broadband systems. Here, by globally optimizing the phase profiles in the visible, we designed and numerically demonstrated a moiré lens based zoom metalens doublet that can achromatically work in the band of 440-640 nm. Such a doublet can achieve a continuous zoom range from 1× to 10×, while also maintaining a high focusing efficiency up to 86.5% and polarization insensitivity.

LncRNA RMST Regulates Neuronal Apoptosis and Inflammatory Response via Sponging miR-150-5p in Parkinson's Disease.

INTRODUCTION: LncRNA rhabdomyosarcoma 2-associated transcript (RMST) serves as a key regulator in neural stem cell fate and is involved in the progression of different neurological diseases. In this research, the serum level and clinical value of RMST in Parkinson's disease (PD) patients were detected, and the underlying mechanism was explored. METHODS: Ninety-nine PD patients and 93 healthy individuals were collected for clinical experiments. SH-SY5Y cells were treated with the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) to establish PD cell models. qRT-PCR was used for the detection of mRNA levels. CCK-8 and flow cytometry were used to detect neuronal viability and apoptosis. The target relationship of RMST with miR-15a-5p was confirmed applying luciferase reporter assay. RESULTS: RMST was present at high levels in both serum of PD patients and PD cell models. Serum RMST had a certain clinical value for the diagnosis of PD with the AUC of 0.892 at a cutoff value of 1.225. Serum RMST was positively associated with the levels of TNF-α (r = 0.421, p < 0.001) and IL-1ß (r = 0.567, p < 0.001) in PD patients. Knockdown of RMST alleviated the apoptosis and inflammatory response of SH-SY5Y cells induced by MPP+. miR-150-5p was the target gene of RMST and less expressed in the clinical serum samples and PD cell models. CONCLUSION: Serum RMST serves as a promising biomarker for the diagnosis of PD. RMST downregulation may regulate the occurrence and development of PD through inhibiting neuron cell apoptosis and the release of inflammatory cytokines via targeting miR-150-5p.

Ectoine production with indigenous Marinococcus sp. MAR2 isolated from the marine environment.

Ectoine has fostered the development of products for skin care and cosmetics. In this study, we employed the marine bacterial strain Marinococcus sp. MAR2 to increase ectoine production by optimizing medium constituents using Response Surface Methodology (RSM) and a fed-batch strategy. The results from the steepest ascent and central composite design indicated that 54 g/L of yeast extract, 14.0 g/L of ammonium acetate, 74.4 g/L of sodium glutamate, and 6.2 g/L of sodium citrate constituted the optimal medium with maximum ectoine production (3.5 g/L). In addition, we performed fed-batch culture in the bioreactor, combining pH and dissolved oxygen to produce ectoine by Marinococcus sp. MAR2. The ectoine production, content, and productivity of 5.6 g/L, 10%, and 3.9 g/L/day were further reached by a fed-batch culture. Thus, the ectoine production by Marinococcus sp. MAR2 using RSM and fed-batch strategy shows its potential for industrial production.

Molecular heterogeneity of non-small cell lung carcinoma patient-derived xenografts closely reflect their primary tumors.

Availability of lung cancer models that closely mimic human tumors remains a significant gap in cancer research, as tumor cell lines and mouse models may not recapitulate the spectrum of lung cancer heterogeneity seen in patients. We aimed to establish a patient-derived tumor xenograft (PDX) resource from surgically resected non-small cell lung cancer (NSCLC). Fresh tumor tissue from surgical resection was implanted and grown in the subcutaneous pocket of non-obese severe combined immune deficient (NOD SCID) gamma mice. Subsequent passages were in NOD SCID mice. A subset of matched patient and PDX tumors and non-neoplastic lung tissues were profiled by whole exome sequencing, single nucleotide polymorphism (SNP) and methylation arrays, and phosphotyrosine (pY)-proteome by mass spectrometry. The data were compared to published NSCLC datasets of NSCLC primary and cell lines. 127 stable PDXs were established from 441 lung carcinomas representing all major histological subtypes: 52 adenocarcinomas, 62 squamous cell carcinomas, one adeno-squamous carcinoma, five sarcomatoid carcinomas, five large cell neuroendocrine carcinomas, and two small cell lung cancers. Somatic mutations, gene copy number and expression profiles, and pY-proteome landscape of 36 PDXs showed greater similarity with patient tumors than with established cell lines. Novel somatic mutations on cancer associated genes were identified but only in PDXs, likely due to selective clonal growth in the PDXs that allows detection of these low allelic frequency mutations. The results provide the strongest evidence yet that PDXs established from lung cancers closely mimic the characteristics of patient primary tumors.

Integrin beta and receptor for activated protein kinase C are involved in the cell entry of Bombyx mori cypovirus.

Receptor-mediated endocytosis using a ß1 integrin-dependent internalization was considered as the primary mechanism for the initiation of mammalian reovirus infection. Bombyx mori cypovirus (BmCPV) is a member of Reoviridae family which mainly infects the midgut epithelium of silkworm; the cell entry of BmCPV is poorly explored. In this study, co-immunoprecipitation (Co-IP), virus overlay protein binding assay (VOPBA), and BmCPV-protein interaction on the polyvinylidene difluoride membrane (BmCPV-PI-PVDF) methods were employed to screen the interacting proteins of BmCPV, and several proteins including integrin beta and receptor for activated protein kinase C (RACK1) were identified as the candidate interacting proteins for establishing the infection of BmCPV. The infectivity of BmCPV was investigated in vivo and in vitro by RNA interference (RNAi) and antibody blocking methods, and the results showed that the infectivity of BmCPV was significantly reduced by either small interfering RNA-mediated silencing of integrin beta and RACK1 or antibody blocking of integrin beta and RACK1. The expression level of integrin beta or RACK1 is not the highest in the silkworm midgut which is a principal target tissue of BmCPV, suggesting that the molecules other than integrin beta or RACK1 might play a key role in determining the tissue tropism of BmCPV infection. The establishment of BmCPV infection depends on other factors, and these factors interacted with integrin beta and RACK1 to form receptor complex for the cell entry of BmCPV.

Proteomic profiles of human lung adeno and squamous cell carcinoma using super-SILAC and label-free quantification approaches.

Nonsmall cell lung cancer (NSCLC) accounts for 85% of lung cancers, and is subdivided into two major histological subtypes: adenocarcinoma (ADC) and squamous cell carcinoma (SCC). There is an unmet need to further subdivide NSCLC according to distinctive molecular features that may be associated with responsiveness to therapies. Four primary tumor-derived xenograft proteomes (two-each ADC and SCC) were quantitatively compared by using a super-SILAC labeling approach together with ultrahigh-resolution MS. Proteins highly differentially expressed in the two subtypes were identified, including 30 that were validated in an independent cohort of 12 NSCLC primary tumor-derived xenograft tumors whose proteomes were quantified by an alternative, label-free shotgun MS methodology. The 30-protein signature contains metabolism enzymes including phosphoglycerate dehydrogenase, which is more highly expressed in SCC, as well as a comprehensive set of cytokeratins and other components of the epithelial barrier, which is therefore distinctly different between ADC and SCC. These results demonstrate the utility of the super-SILAC method for the characterization of primary tissues, and compatibility with datasets derived from different MS-based platforms. The validation of proteome signatures of NSCLC subtypes supports the further development and application of MS-based quantitative proteomics as a basis for precision classifications and treatments of tumors. All MS data have been deposited in the ProteomeXchange with identifier PXD000438 (http://proteomecentral.proteomexchange.org/dataset/PXD000438).

Daptomycin-loaded polymethylmethacrylate bone cement for joint arthroplasty surgery.

Antibiotic-loaded acrylic bone cement has been frequently used as an infection prophylaxis or antibiotic-loaded spacer in infected arthroplasty. In addition, daptomycin has been used recently against broad spectrum Gram-positive organisms. The goal of this in vitro study is to investigate the bacteriacidal and mechanical properties of daptomycin-incorporated polymethylmethacrylate (PMMA) bone cement and evaluate its feasibility for clinical use. Daptomycin (0.5, 1, or 2 g) was premixed with 40 g of PMMA bone cement powder before curing. The mechanical properties of the daptomycin-loaded acrylic bone cement (DLABC) were estimated following standard guidance, and the release profile and kinetics of daptomycin from PMMA were analyzed. The antimicrobial efficacy of DLABC was determined with a zone of inhibition (ZOI) assay against Staphylococcus aureus, Staphylococcus epidermis, Enterococcus faecalis, and Enterococcus faecium, respectively. The results showed that the compressive strength, of PMMA bone cement, which was higher than 100 MPa in all groups, was sufficient according to ISO 5833 after incorporation of daptomycin. The encapsulated daptomycin was released for 2 weeks with a 9.59 ± 0.85%, 15.25 ± 0.69%, and 20.64 ± 20.33% released percentage on the first day in the low, mid, and high groups, respectively. According to the calculated release kinetics, incorporated daptomycin should be 3.3 times the original dose to double its release. Although all recipes of DLABC had a microbial inhibitory effect, the effect with a higher encapsulated amount of daptomycin was more significant. Therefore, we believe that daptomycin can be locally delivered from PMMA bone cement at the surgical site as a prophylactic or treatment for osteomyelitis against Gram-positive organisms with intact cement function.

A Large-Scale and Low-Cost Thermoacoustic Loudspeaker Based on Three-Dimensional Graphene Foam.

Graphene is a promising material for thermoacoustic sources due to its extremely low heat capacity per unit area and high thermal conductivity. However, current graphene thermoacoustic devices have limited device area and relatively high cost, which limit their applications of daily use. Here, we adopt a dip-coating method to fabricate a large-scale and cost-effective graphene sound source. This sound source has the three-dimensional (3D) porous structure that can increase the contact area between graphene and air, thus assisting heat to release into the air. In this method, polyurethane (PU) is used as a support, and graphene nanoplates are attached onto the PU skeleton so that a highly flexible graphene foam (GrF) device is obtained. At a measuring distance of 1 mm, it can emit sound at up to 70 dB under the normalized input power of 1 W. Considering its unique porous structure, we establish a thermoacoustic analysis model to simulate the acoustic performance of GrF. Furthermore, the obtained GrF can be made up to 44 in. (100 cm × 50 cm) in size, and it has good flexibility and processability, which broadens the application fields of GrF loudspeakers. It can be attached to the surfaces of objects with different shapes, making it suitable to be used as a large-area speaker in automobiles, houses, and other application scenarios, such as neck mounted speaker. In addition, it can also be widely used as a fully flexible in-ear earphone.

Age-, season- and gender-specific reference intervals of serum 25-hydroxyvitamin D3 for healthy children (0 ~ 18 years old) in Nanning area of China.

Establishing specific reference intervals (RIs) of serum 25-hydroxyvitamin D3 [25(OH)D] for children is essential for improving the accuracy of diagnosis and prognosis monitoring of diseases such as rickets and growth retardation. The study including 6,627 healthy children was conducted to establish specific RIs of 25(OH)D for children in Nanning area of China. The results showed that there were statistically significant differences among age, season, and gender of serum 25(OH)D levels, and the age-specific RIs of serum 25(OH)D were 20.3 ~ 53.6 ng/mL for 0 ~ ≤ 1 year and 18.9 ~ 49.6 ng/mL for 2 ~ ≤ 3 years. The age-, season-specific RIs of serum 25(OH)D for 4 ~ ≤ 6 years in spring-summer and autumn-winter were 15.8 ~ 42.6 ng/mL and 15.2 ~ 37.7 ng/mL, respectively. The age-, gender-specific RIs of serum 25(OH)D for 7 ~ ≤ 18 years for males and females were 12.1 ~ 36.1 ng/mL and 10.8 ~ 35.3 ng/mL, respectively. This study successfully established the RIs of serum 25(OH)D, which may help to improve disease diagnosis and monitoring for children in the Nanning area of China.

Single-cell spatial landscape of immunotherapy response reveals mechanisms of CXCL13 enhanced antitumor immunity.

BACKGROUND: Immunotherapy has revolutionized clinical outcomes for patients suffering from lung cancer, yet relatively few patients sustain long-term durable responses. Recent studies have demonstrated that the tumor immune microenvironment fosters tumorous heterogeneity and mediates both disease progression and response to immune checkpoint inhibitors (ICI). As such, there is an unmet need to elucidate the spatially defined single-cell landscape of the lung cancer microenvironment to understand the mechanisms of disease progression and identify biomarkers of response to ICI. METHODS: Here, in this study, we applied imaging mass cytometry to characterize the tumor and immunological landscape of immunotherapy response in non-small cell lung cancer by describing activated cell states, cellular interactions and neighborhoods associated with improved efficacy. We functionally validated our findings using preclinical mouse models of cancer treated with anti-programmed cell death protein-1 (PD-1) immune checkpoint blockade. RESULTS: We resolved 114,524 single cells in 27 patients treated with ICI, enabling spatial resolution of immune lineages and activation states with distinct clinical outcomes. We demonstrated that CXCL13 expression is associated with ICI efficacy in patients, and that recombinant CXCL13 potentiates anti-PD-1 response in vivo in association with increased antigen experienced T cell subsets and reduced CCR2+ monocytes. DISCUSSION: Our results provide a high-resolution molecular resource and illustrate the importance of major immune lineages as well as their functional substates in understanding the role of the tumor immune microenvironment in response to ICIs.

Epigenetic plasticity cooperates with cell-cell interactions to direct pancreatic tumorigenesis.

The response to tumor-initiating inflammatory and genetic insults can vary among morphologically indistinguishable cells, suggesting as yet uncharacterized roles for epigenetic plasticity during early neoplasia. To investigate the origins and impact of such plasticity, we performed single-cell analyses on normal, inflamed, premalignant, and malignant tissues in autochthonous models of pancreatic cancer. We reproducibly identified heterogeneous cell states that are primed for diverse, late-emerging neoplastic fates and linked these to chromatin remodeling at cell-cell communication loci. Using an inference approach, we revealed signaling gene modules and tissue-level cross-talk, including a neoplasia-driving feedback loop between discrete epithelial and immune cell populations that was functionally validated in mice. Our results uncover a neoplasia-specific tissue-remodeling program that may be exploited for pancreatic cancer interception.

Alternative splicing, expression patterns and promoter characters of vasa-like gene from the silkworm, Bombyx mori.

VASA is considered to be one of the most reliable molecular marker of germ cells. In order to study the Bombyx mori vasa-like gene (Bmvlg), the cDNAs of Bmvlg were cloned and sequenced, and the results showed that the Bmvlg gene from the fifth instar larval testes had four alternative splicing isoforms. The open reading frame (ORF) of the longest isoform was composed of 1,806 nucleotides encoding 601 amino acid residues and contained some known conserved domains. The other three isoforms had complete ORF, suggesting that the Bmvlg gene had several alternative splicing forms, completely different from that of Drosophila melanogaster. The results of sequencing demonstrated that the Bmvlg gene promoter had several elements conserved in eukaryotic and gonadal tissue-specific promoters. To detect the specificity of the Bmvlg promoter, a transient expression vector pSK-vlg-DsRed-polyA with a red fluorescent protein gene (DsRed), controlled by the Bmvlg promoter and a vector pIZT/V5-His-vlg-DsRed containing a Bmvlg fused with DsRed driven by the Bmvlg promoter, was constructed, respectively. Red fluorescence could be observed in some transfected BmN cells derived from silkworm ovaries and in the eggs injected with the vector pSK-vlg-DsRed-polyA, but red fluorescence could not be detected in the tissues of silkworm larva, after the transient expression vector was injected into blood, suggesting the Bmvlg promoter had gonadal tissue specificity. The transcription levels of Bmvlg in gonads of the fourth and fifth instar larvae were determined by fluorescent quantitative PCR, and the results revealed that the expression level of the Bmvlg gene in testes was slightly higher than that in ovaries. The expression levels of Bmvlg were lower in the fourth instar larva than that in the fifth instar larvae. Moreover, subcellular localization experiments showed that Bmvlg mainly existed in cytoplasm. These results provided new clues for understanding the function of the Bmvlg gene.

From Materials to Devices: Graphene toward Practical Applications.

Graphene, as an emerging 2D material, has been playing an important role in flexible electronics since its discovery in 2004. The representative fabrication methods of graphene include mechanical exfoliation, liquid-phase exfoliation, chemical vapor deposition, redox reaction, etc. Based on its excellent mechanical, electrical, thermo-acoustical, optical, and other properties, graphene has made a great progress in the development of mechanical sensors, microphone, sound source, electrophysiological detection, solar cells, synaptic transistors, light-emitting devices, and so on. In different application fields, large-scale, low-cost, high-quality, and excellent performance are important factors that limit the industrialization development of graphene. Therefore, laser scribing technology, roll-to-roll technology is used to reduce the cost. High-quality graphene can be obtained through chemical vapor deposition processes. The performance can be improved through the design of structure of the devices, and the homogeneity and stability of devices can be achieved by mechanized machining means. In total, graphene devices show promising prospect for the practical fields of sports monitoring, health detection, voice recognition, energy, etc. There is a hot issue for industry to create and maintain the market competitiveness of graphene products through increasing its versatility and killer application fields.

A protein containing the DUF1471 domain regulates biofilm formation and capsule production in Klebsiella pneumoniae.

BACKGROUND/PURPOSE: Biofilms formed by Klebsiella pneumoniae on medical devices increase infection risk. Fimbriae and capsule polysaccharides (CPSs) are important factors involved in biofilm formation. KP1_4563 in K. pneumoniae NTUH-K2044, a small protein containing the DUF1471 domain, was reported to inhibit type 3 fimbriae function. In this study, we aimed to determine whether the KP1_4563 homolog is conserved in each K. pneumoniae isolate and what role it has in Klebsiella biofilms. METHODS: The genomes of K. pneumoniae NTUH-K2044, CG43, MGH78578, KPPR1 and STU1 were compared. The KP1_4563 homolog in K. pneumoniae STU1 was named orfX. Biofilms of wild-type and orfX mutant strains from K. pneumoniae STU1 and one clinical isolate, 83535, were quantified. Transcription levels of the type 3 fimbrial genes, mrkA and mrkH, were investigated by RT-qPCR. MrkA of the wild-type and orfX mutant were observed by Western blotting. The morphology of bacterial cells was observed by transmission electron microscopy (TEM). Bacterial CPSs were quantified. RESULTS: The gene and upstream region of orfX were conserved among the five K. pneumoniae isolates. Deletion of orfX enhanced Klebsiella biofilm formation. However, the amount of mRNA from mrkA and mrkH and the level of MrkA protein were not different between the wild type and orfX mutant. In contrast, the amount of CPS in orfX mutants was increased, compared to their parental strains, STU1 and 83535. CONCLUSION: The role of orfX is speculated to be conserved in most K. pneumoniae isolates. OrfX negatively controlled biofilm formation by reducing CPS, not type 3 fimbriae, production.

Spatially mapping the immune landscape of melanoma using imaging mass cytometry.

Melanoma is an immunogenic cancer with a high response rate to immune checkpoint inhibitors (ICIs). It harbors a high mutation burden compared with other cancers and, as a result, has abundant tumor-infiltrating lymphocytes (TILs) within its microenvironment. However, understanding the complex interplay between the stroma, tumor cells, and distinct TIL subsets remains a substantial challenge in immune oncology. To properly study this interplay, quantifying spatial relationships of multiple cell types within the tumor microenvironment is crucial. To address this, we used cytometry time-of-flight (CyTOF) imaging mass cytometry (IMC) to simultaneously quantify the expression of 35 protein markers, characterizing the microenvironment of 5 benign nevi and 67 melanomas. We profiled more than 220,000 individual cells to identify melanoma, lymphocyte subsets, macrophage/monocyte, and stromal cell populations, allowing for in-depth spatial quantification of the melanoma microenvironment. We found that within pretreatment melanomas, the abundance of proliferating antigen-experienced cytotoxic T cells (CD8+CD45RO+Ki67+) and the proximity of antigen-experienced cytotoxic T cells to melanoma cells were associated with positive response to ICIs. Our study highlights the potential of multiplexed single-cell technology to quantify spatial cell-cell interactions within the tumor microenvironment to understand immune therapy responses.

Primary tumor xenografts of human lung adeno and squamous cell carcinoma express distinct proteomic signatures.

Nonsmall cell lung carcinoma (NSCLC) accounts for 80% of lung cancers. The most prevalent subtypes of NSCLC are adenocarcinoma (ADC) and squamous cell carcinoma (SCC), which combined account for approximately 90%. Ten resected NSCLC patient tumors (5 ADC and 5 SCC) were directly introduced into severely immune deficient (NOD-SCID) mice, and the resulting xenograft tumors were analyzed by standard histology and immunohistochemistry (IHC) and by proteomics profiling. Mass spectrometry (MS) methods involving 1- and 2-dimensional LC-MS/MS, and multiplexed selective reaction monitoring (SRM, or MRM), were applied to identify and quantify the xenograft proteomes. Hierarchical clustering of protein profiles distinguished between the ADC and SCC subtypes. The differential expression of 178 proteins, including a comprehensive panel of intermediate filament keratin proteins, was found to constitute a distinctive proteomic signature associated with the NSCLC subtypes. Epidermal growth factor receptor (EGFR) was expressed in ADC and SCC xenografts, and EGFR network activation was assessed by phosphotyrosine profiling by Western blot analysis and SRM measurement of EGFR levels, and mutation analysis. A multiplexed SRM/MRM method provided relative quantification of several keratin proteins, EGFR and plakophilin-1 in single LC-MS/MS runs. The protein quantifications by SRM and MS/MS spectral counting were associated with superior dynamic range and reproducibility but were otherwise consistent with orthogonal methods including IHC and Western immuno blotting. These findings illustrate the potential to develop a comprehensive MS-based platform in oncologic pathology for better classification and potentially treatment of NSCLC patients.

Biodegradable and biocompatible biomaterial, polyhydroxybutyrate, produced by an indigenous Vibrio sp. BM-1 isolated from marine environment.

Polyhydroxybutyrate (PHB) is one of the polyhydroxyalkanoates (PHAs) which has biodegradable and biocompatible properties. They are adopted in the biomedical field, in, for example, medical implants and drug delivery carriers. This study seeks to promote the production of PHB by Vibrio sp. BM-1, isolated from a marine environment by improving constituents of medium and implementing an appropriate fermentation strategy. This study successfully developed a glycerol-yeast extract-tryptone (GYT) medium that can facilitate the growth of Vibrio sp. BM-1 and lead to the production of 1.4 g/L PHB at 20 h cultivation. This study also shows that 1.57 g/L PHB concentration and 16% PHB content were achieved, respectively, when Vibrio sp. BM-1 was cultivated with MS-GYT medium (mineral salts-supplemented GYT medium) for 12 h. Both cell dry weight (CDW) and residual CDW remained constant at around 8.2 g/L and 8.0 g/L after the 12 h of cultivation, until the end of the experiment. However, both 16% of PHB content and 1.57 g/L of PHB production decreased rapidly to 3% and 0.25 g/L, respectively from 12 h of cultivation to 40 h of cultivation. The results suggest that the secretion of PHB depolymerase that might be caused by the addition of mineral salts reduced PHB after 12 h of cultivation. However, work will be done to explain the effect of adding mineral salts on the production of PHB by Vibrio sp. BM-1 in the near future.