Electronic field effect detection of SARS-CoV-2 N-protein before the onset of symptoms.

As part of the efforts to contain the pandemic, researchers around the world have raced to develop testing platforms to detect the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the Coronavirus disease 2019 (COVID-19). Within the different detection platforms studied, the field effect transistor (FET) is a promising device due to its high sensitivity and fast detection capabilities. In this work, a graphene-based FET which uses a boron and nitrogen co-doped graphene oxide gel (BN-GO gel) transducer functionalized with nucleoprotein antibodies, has been investigated for the detection of SARS-CoV-2 nucleocapsid (N)-protein in buffer. This biosensor was able to detect the viral protein in less than 4 min, with a limit of detection (LOD) as low as 10 ag/mL and a wide linear detection range stretching over 11 orders of magnitude from 10 ag/mL-1 µg/mL. This represents the lowest LOD and widest detection range of any COVID-19 sensor and thus can potentially enable the detection of infected individuals before they become contagious. In addition to its potential use in the COVID-19 pandemic, our device serves as a proof-of-concept of the ability of functionalized BN-GO gel FETs to be used for ultrasensitive yet robust biosensors.

CUB and zona pellucida-like domain-containing protein 1 (CUZD1): a novel serological biomarker for ovarian cancer.

OBJECTIVES: To measure the levels of serum CUB and zona pellucida-like domain-containing protein 1 (CUZD1) in patients with ovarian cancer (OvCa), benign gynecological conditions and healthy women and in a number of other cancer types (breast, colorectal, lung, prostate and testicular). DESIGN AND METHODS: Serum CUZD1 levels were measured with a commercial enzyme-linked immunosorbent assay (ELISA). All specimens were analyzed in duplicate. Preliminary verification was performed in serum using 9 healthy women and 20 late stage (III-IV) OvCa patients. An independent cohort of serum samples was used to validate the verification results (18 late stage OvCa, 8 benign gynecological conditions and 8 healthy controls). The following specimens were used for the other cancer types of unknown stage-breast (n=11), colorectal (n=10), lung (n=10), prostate (n=15) and testicular (n=10). RESULTS: Serum CUZD1 was significantly elevated in ovarian cancer patients (range 95-668 µg/L) as compared to healthy controls (range 0.7-2.5 µg/L). The independent cohort of OvCa samples confirmed the preliminary verification results. CUZD1 was also elevated in breast and lung cancer specimens and not in colorectal, prostate and testicular cancer specimens. CONCLUSIONS: CUZD1 appears to be a highly promising novel serum biomarker for OvCa diagnosis. Its performance in the 2 independent cohorts examined, and in lung and breast cancer patients warrants further investigation.

Impact of expression differences of kallikrein-related peptidases and of uPA and PAI-1 between primary tumor and omentum metastasis in advanced ovarian cancer.

BACKGROUND: Primary tumor levels of serine proteases of the kallikrein-related peptidases (KLK) family as well as urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 impact disease course in ovarian cancer. The changes in levels of these factors from primary tumor to omentum metastasis ('level differentials') could thus be associated with metastastic processes. PATIENTS AND METHODS: Protein levels of seven tissue KLK (KLK5-8, 10, 11, 13), uPA, and PAI-1 were determined in extracts of primary tumor tissue and corresponding omentum metastasis of 54 ovarian cancer patients. RESULTS: Higher level differentials of KLK5-8, 10-11, and uPA were associated with residual tumor >10 mm. Residual tumor and larger level differentials of KLK5-7, 10, and uPA were associated with disease progression in the whole cohort. Remarkably, level differentials of KLK5-8 and 10-11 strongly impacted disease progression even in patients with residual tumor mass ≤10 mm; hence, the observed impact of level differentials in KLK5-7 and 10 on disease progression was not simply attributable to their association with surgical success. CONCLUSION: Since they impact both surgical outcome and survival in advanced ovarian cancer, measurement of level differentials could support clinical decisions on surgical and systemic therapy or help in patient selection for novel targeted therapies.

The use of kallikrein-related peptidases as adjuvant prognostic markers in colorectal cancer.

Several members of the human tissue kallikrein-related peptidase (KLK) family are emerging cancer biomarkers. The aim of this study was to analyse the expression of a panel of KLKs in colorectal cancer and to find out if the multiparametric combination of them can increase the accuracy of prediction of patients survival beyond the traditional clinical information. Nine KLKs (KLK5-8, KLK10, KLK11, KLK13-15) were measured using ELISA assays in cytosolic extracts of 122 colon cancer tissues and their nearby normal mucosa, obtained during surgery. The mean levels of almost all KLKs in tumour tissues were significantly different from their counterparts of normal tissue (P<0.0001). KLK 5, 6, 7, 13, 14 were significantly associated with overall survival in univariate analysis, but after adjusting for age, TNM and differentiation stage, only KLK5 (HR: 1.24 (95% CI: 1.05-1.47)), KLK7 (HR: 1.57 (95% CI: 1.04-2.37)) and KLK14 (HR: 1.43 (95% CI: 1.05-1.94)) remained significant. Addition of a panel of selected KLK markers to clinical parameters gave an increment in AUC of 0.86 beyond the clinical factors at year 1, showing that it can increase the accuracy of prediction of overall survival beyond the traditional clinical information, particularly the short-term (1 year) survival after surgery.

Kallikrein gene downregulation in breast cancer.

Recent evidence suggests that many members of the human kallikrein gene family are differentially regulated in breast cancer and other endocrine-related malignancies. In this study, we utilised the serial analysis of gene expression (SAGE) and expressed sequence tag (EST) databases of the Cancer Genome Anatomy Project (CGAP) to perform in silico analyses of the expression pattern of the 15 human kallikrein genes in normal and cancerous breast tissues and cell lines using different analytical tools such as Virtual Northern blotting, Digital Differential Display and X-profiler. Our results indicate that at least four kallikrein genes (KLK5, 6, 8, 10) are downregulated in breast cancer. Probing eight normal and 24 breast cancer SAGE libraries with gene-specific tags for each of the above kallikreins indicated moderate-to-high expression densities in normal breast (27-319 tags per million; tpm, in two to five out of eight libraries), compared to no or low expression (0 - 34 tpm in zero to two libraries out of 24) in breast cancer. These data were verified by screening the EST databases, where all mRNA clones isolated for these genes, except for one in each, were from normal breast libraries, with no clones detected from breast cancer tissues or cell lines (with the exception of KLK8). X-profiler comparison of two pools of normal and breast cancer libraries further verified the presence of significant downregulation of expression levels of 4 of the kallikreins genes (KLK5, 6, 10, 12). We experimentally verified the downregulation of these four kallikreins (KLK5, 6, 8, 10 and 12) by RT - PCR analysis.

Identification of heat shock protein 90 and other proteins as tumour antigens by serological screening of an ovarian carcinoma expression library.

Serological screening of recombinant cDNA expression libraries has been widely used for the identification of tumour antigens in various cancer types. Identification of tumour antigens in ovarian cancer may facilitate the development of vaccine-based therapies and of disease biomarkers. The purpose of our investigation is to identify tumour antigens in ovarian cancer by using the serological analysis of recombinant cDNA expression libraries method. A recombinant ovarian carcinoma cDNA expression library was screened with ascites fluid, pooled from five ovarian cancer patients. Twelve tumour antigens encoded by known genes were isolated, including ribosomal protein S18, heat shock protein 90, JK-recombination signal binding protein, ribonucleoprotein H1, RAN binding protein 7, TG-interacting factor, eukaryotic translation initiation factor p40 subunit, human amyloid precursor protein-binding protein 1, ribosomal protein L8, CDC23, IQ motif containing GTPase activating protein 1, and ribosomal protein L3. Heat shock protein 90 was chosen for further investigation. The prevalence of hsp90 autoantibodies in ovarian cancer was determined with immunoassay. Sera from 22 normal females, 32 from ovarian cancer (22 stage III/IV, 10 stage I/II), 37 colorectal cancer, 13 breast cancer, 10 lung cancer, 20 benign gynaecologic diseases, and 10 benign breast lesions were screened. Seven (32%) stage III/IV ovarian cancer, 1 (10%) stage I/II ovarian cancer, 1 (3%) colorectal cancer, 1 (8%) breast cancer, and 1 (5%) benign gynaecologic disease sera were found to contain hsp90 autoantibodies. These data support the view that hsp90 autoantibodies are frequently found in late stage ovarian cancer. Hsp90 may, therefore, represent a novel biomarker for ovarian cancer and a candidate ovarian cancer vaccine target.

Higher expression of human kallikrein 10 in breast cancer tissue predicts tamoxifen resistance.

The human tissue kallikreins are secreted serine proteases, encoded by a group of homologous genes clustered in tandem on chromosome 19q13.3-4. Human kallikrein 6 and human kallikrein 10 are two new members of this family. Recently, we developed highly sensitive and specific immunofluorometric assays for human kallikrein 6 and human kallikrein 10, which allow for their quantification in tissue extracts and biological fluids. Both human kallikrein 6 and human kallikrein 10 are found to be down-regulated in breast cancer cell lines, suggesting that they may be involved in breast cancer pathogenesis and progression. In this study, we investigated the potential value of human kallikrein 6 and human kallikrein 10 as prognostic and predictive factors in breast cancer. We quantified human kallikrein 6 and human kallikrein 10 protein levels in 749 breast tumour cytosolic extracts and correlated this data with various clinicopathological variables and patient outcomes. Human kallikrein 6 and human kallikrein 10 are positively correlated with each other. Higher human kallikrein 6 and human kallikrein 10 protein levels are associated with younger age, pre-menopausal, status and tumours which are negative for oestrogen and progesterone receptors. No correlation was found between human kallikrein 6 and human kallikrein 10 levels and tumour size, grade, and nodal status. Survival analysis showed that neither human kallikrein 6 nor human kallikrein 10 are related to the rate of relapse-free and overall survival. In the analysis with respect to response to tamoxifen therapy, although human kallikrein 6 levels were not associated with tamoxifen responsiveness, higher levels of human kallikrein 10 were significantly associated with a poor response rate. This association remained significant in the multivariate analysis. Furthermore, higher human kallikrein 10 levels were significantly related with a short progression-free and post-relapse overall survival after start of tamoxifen treatment for advanced disease. Taken together, our results suggest that although human kallikrein 6 and human kallikrein 10 are not prognostic markers for breast cancer, human kallikrein 10 is an independent predictive marker for response of tamoxifen therapy.

Molecular cloning of a novel human gene on chromosome 4p11 by immunoscreening of an ovarian carcinoma cDNA library.

In our efforts to identify immunoreactive antigens in ovarian cancer, we used the method of immunoscreening of an ovarian carcinoma cDNA expression library with ascites fluid from ovarian cancer patients. Among many positive clones, one was found to contain partial sequence of a novel gene. By searching expressed sequence tags (ESTs) and human genome project databases as well as by screening other cDNA libraries and by RT-PCR strategies, we were able to obtain the full-length cDNA sequence (1.4 kb) and establish the genomic organization of this new gene. We also identified two alternatively spliced forms, encoding for slightly different proteins. The longer form (1.4 kb) is predicted to encode for a 27.6 kDa protein of 245 amino acids. The shorter form (1.3 kb) encodes for a truncated protein of 20.7 kDa and 208 amino acids. These proteins are not significantly homologous to any known protein in the GenBank database. This gene is composed of nine exons and eight introns. By fluorescence in situ hybridization (FISH), it was mapped to chromosome 4p11. This gene is highly expressed in many tissues, including testis, brain, placenta, ovary, prostate, and mammary gland. The high level expression of the shorter form is restricted to the central nervous system, including brain, cerebellum, and spinal cord, suggesting that this form may have a unique function in the central nervous system.

Human kallikrein 6 (zyme/protease M/neurosin): a new serum biomarker of ovarian carcinoma.

BACKGROUND: There is an urgent need for discovery and validation of new serum biomarkers for ovarian carcinoma. Early diagnosis of ovarian cancer with serologic analysis may improve clinical outcomes through administration of effective treatment. Human kallikrein 6 (hK6, encoded by the KLK6 gene) is a serine protease of the kallikrein gene family. Recently, we were able to develop an immunofluorometric procedure for the quantitative measurement of hK6 in biologic fluids, including serum. METHODS: We have used an hK6-specific immunofluorometric assay to quantify hK6 protein in a large number of serum samples from normal individuals, as well as from patients with various malignancies. RESULTS: We report for the first time, significant increase of serum hK6 concentration in a large proportion of patients with ovarian carcinoma. The elevations of hK6 appear to be relatively specific for ovarian cancer because other malignancies did not cause any increase in the concentration of this biomarker in serum. Serial hK6 measurements appear to correlate with CA125 levels in patients monitored postsurgery. CONCLUSIONS: This is the first report describing significant elevations of hK6 concentration in serum of ovarian cancer patients. These data suggest that hK6 may represent a potential new biomarker for diagnosis and monitoring of ovarian carcinoma.

Immunofluorometric assay of human kallikrein 6 (zyme/protease M/neurosin) and preliminary clinical applications.

BACKGROUND: The human kallikrein gene family has contributed the best prostatic biomarkers currently available, including prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2). Recently, new members of the human kallikrein gene family have been identified. One new member is the KLK6 gene, encoding for human kallikrein 6 (hK6), which is also known as zyme/protease M/neurosin. In this paper, we describe development of antibodies and a sensitive immunofluorometric procedure for hK6 protein. METHODS: Recombinant hK6 protein was used as immunogen to develop polyclonal antibodies in rabbits and mice. These antibodies were used to develop a sandwich-type time-resolved immunofluorometric procedure for hK6. RESULTS: The newly developed hK6 immunofluorometric assay has a detection limit of 0.5 microg/L and upper concentration range of 200 microg/L. The assay is highly specific (no detectable cross-reactivity from PSA and hK2) and was used to quantify hK6 protein in various biologic fluids. Highest concentrations of hK6 were found in milk of lactating women, cerebral spinal fluid, nipple aspirate fluid, and breast cyst fluid. hK6 was also detected in male and female serum, in the majority of seminal plasmas and in a small fraction of amniotic fluids and breast tumor cytosols. hK6 was not detectable in urine. Chromatographic studies indicated that hK6 is present in these biologic fluids in its free, 30-kDa form. CONCLUSIONS: This is the first reported sensitive immunofluorometric procedure for quantifying hK6 protein. hK6 is a secreted proteolytic enzyme that is found at high levels in cerebrospinal fluid and all breast secretions. This assay will facilitate further studies to examine the possible application of hK6 in diagnostics, including cancer and neurodegenerative disorders.

The KLK7 (PRSS6) gene, encoding for the stratum corneum chymotryptic enzyme is a new member of the human kallikrein gene family - genomic characterization, mapping, tissue expression and hormonal regulation.

The human stratum corneum chymotryptic enzyme (HSCCE; PRSS6, KLK7 gene) has been purified from human stratum corneum and is known to participate in the cell shedding process. The cDNA of the gene has previously been reported. Here, we describe the identification of 5' and 3' extensions of the published mRNA, and the complete genomic organization of the gene. KLK7 is composed of five coding exons which have similar lengths to exons of other kallikrein-like genes. The intron phases are completely conserved between this gene and other members of the kallikrein-like gene family. Precise mapping of KLK7 has indicated that it is located at chromosomal locus 19q13. 3-q13.4 between the already known genes zyme (KLK6) (centromere) and neuropsin (KLK8) (telomere). Until recently, it was thought that this gene is expressed only in the skin. We here provide evidence that KLK7 is also expressed at relatively high levels in the central nervous system, kidney, mammary and salivary glands. Its expression is up-regulated by estrogens and glucocorticoids in the breast carcinoma cell line BT-474. The cDNA and protein of this gene are homologous to sequences of other kallikrein-like genes. The gene encodes for a secreted protein. Phylogenetic analysis, the close structural similarities, and its co-localization in the same chromosomal region, suggest that the gene encoding for the stratum corneum chymotryptic enzyme is a new member of the expanded human kallikrein gene family.

Comparison of the percent free prostate-specific antigen levels in the serum of healthy men and in men with recurrent prostate cancer after radical prostatectomy.

The percentage of free PSA in serum is currently used to better discriminate between patients with prostate cancer and patients with benign prostatic hyperplasia, in prostate cancer screening programs. We measured using non-competitive immunological techniques, the total PSA and free PSA in post-surgical serum of prostate cancer patients who underwent radical prostatectomy and then relapsed. We compared these data with those of a group of 40 age-matched men with no evidence of prostatic disease. Although in general, patients with prostate cancer had lower percentage of free PSA in serum in comparison to the controls, a subset of these patients (approximately 20%) had percent free PSA significantly higher than the levels considered as exclusive of prostate cancer in screening programs. We also found that percent free PSA does not correlate significantly with most of the standard clinical or pathological indicators of prostate cancer aggressiveness. Only a weak negative association with Gleason Score was observed. The percent free PSA in serum of relapsing prostate cancer patients varies within a relatively wide range and does not correlate significantly with indicators of cancer aggressiveness. The use of percent free PSA for excluding prostate cancer in screening programs must be approached with caution until the mechanism of low percent free PSA in the majority but not all prostate cancer patients is elucidated.

Human kallikrein 6 as a biomarker of alzheimer's disease.

OBJECTIVES: Alzheimer's disease (AD) is a major cause of dementia in the elderly. It is generally difficult to diagnose accurately early AD. A few biomarkers, including tau protein and amyloid beta-42, are now used as aids for diagnosis and monitoring of AD. Our aim was to examine the possible use of cerebrospinal fluid, blood and tissue, and human kallikrein 6 (hK6) concentration as a marker of AD. METHODS: We have used a highly sensitive and specific immunofluorometric procedure for measuring hK6. We measured hK6 in tissue extracts from AD brain or normal individuals, in cerebrospinal fluids of AD patients or normals and in whole blood of AD patients and normals and compared the findings. We have used ten pairs of AD/normal controls in all cases. RESULTS: We found that hK6 concentration is tissue extracts from AD brain were approximately twofold lower than extracts from normal controls. Further, we found that cerebrospinal fluid hK6 concentration is approximately a threefold increase, in comparison to cerebrospinal fluid controls (p = 0.001). We have also found that the whole blood hK6 concentration in AD patients is about ten times higher than hK6 concentration in normal controls (p = 0.002). We have immunohistochemically localized the expression of hK6 in epithelial cells of the chorioid plexus. CONCLUSIONS: This is the first report describing significant elevations of cerebrospinal fluid and plasma and whole blood hK6 concentration in AD patients, in comparison to controls. These data suggest that hK6 may constitute a new biomarker for diagnosis and monitoring of AD.