Efficacy of Antiviral Treatment in Hepatitis C Virus (HCV)-Driven Monoclonal Gammopathies Including Myeloma

Multiple myeloma (MM) remains an incurable plasma cell malignancy. While its origin is enigmatic, an association with infectious pathogens including hepatitis C virus (HCV) has been suggested. Here we report nine patients with monoclonal gammopathy of undetermined significance (MGUS) or MM with previous HCV infection, six of whom received antiviral treatment. We studied the evolution of the gammopathy disease, according to anti-HCV treatment and antigen specificity of purified monoclonal immunoglobulin, determined using the INNO-LIA™ HCV Score assay, dot-blot assays, and a multiplex infectious antigen microarray. The monoclonal immunoglobulin from 6/9 patients reacted against HCV. Four of these patients received antiviral treatment and had a better evolution than untreated patients. Following antiviral treatment, one patient with MM in third relapse achieved complete remission with minimal residual disease negativity. For two patients who did not receive antiviral treatment, disease progressed. For the two patients whose monoclonal immunoglobulin did not react against HCV, antiviral treatment was not effective for MGUS or MM disease. Our results suggest a causal relationship between HCV infection and MGUS and MM progression. When HCV was eliminated, chronic antigen-stimulation disappeared, allowing control of clonal plasma cells. This opens new possibilities of treatment for MGUS and myeloma.

A Ponceau S Staining-Based Dot Blot Assay for Rapid Protein Quantification of Biological Samples

Despite the availability of a wide range of commercial kits, protein quantification is often unreliable, especially for tissue-derived samples, leading to uneven loading in subsequent experiments. Here we show that the widely used Bicinchoninic Acid (BCA) assay tends to underestimate protein concentrations of tissue samples. We present a Ponceau S staining-based dot-blot assay as an alternative for protein quantification. This method is simple, rapid, more reliable than the BCA assay, compatible with biological samples lysed in RIPA or 2x SDS gel-loading buffer, and also inexpensive.

Dot blot immunochemical and infrared analyses of the adhesive layer applied to the painting Imago Pietatis by Domenico Morone

Purpose To investigate the nature of the materials used in the adhesive layer of the Imago Pietatis painting (end of the fifteenth century—beginning of the sixteenth century) by Domenico Morone as a prerequisite for its restoration. Methods Micro-FTIR spectra of the animal glue and a polished cross-section were acquired by a Jasco IRT3000 spectrometer, equipped with a 32× Cassegrain objective. A dot blot immunoassay was used to characterise a minor component of the adhesive layer. Results Micro-FTIR was used as an effective diagnostic tool to detect the major component of the adhesive layer and the binder of the paint. Despite the ageing, the complex matrix and the micro-size of the sample, using a dot blot immunoassay, it was possible to quantify 3.7 ± 2.0 ng of ovalbumin per microgram of sample (corresponding to 0.004 ± 0.002% of the weight). Conclusions The findings were in line with conservation practices described in the old treatises, confirming the correct interpretation of the adhesive layer compounds added to the painting and suggesting for the cleaning the use of an anionic water-soluble surfactant highly effective in the removing of proteinaceous materials.

Reverse dot blot hybridization assay— An expeditious diagnostic tool for drug resistant TB

Tuberculosis (TB) is one of the leading infectious diseases that is highly transmissible. It is reported to cause approximately 10 million infections and 1.4 million deaths globally in the year of 2019.¹ Prompt detection and treatment of TB are essential to intercept the proliferation of the disease. In the case of drug- resistant TB, failure to detect the resistance of anti TB drugs may give rise to extensively drug- resistant tuberculosis (XDR-TB). Thus, the rapid detection of drug resistance is vital;, however, the current standard drug susceptibility tests (DST) may take up to 12 weeks raising an alarming concern.² A study was done in China by Li Wan et al, on the accuracy of the reverse dot blot hybridization assay (RDBH) for rapidly detecting the resistance of four anti TB drugs (rifampicin (RIF), isoniazid (INH), streptomycin (SM) and ethambutol (EMB)) in mycobacterium tuberculosis (MTB) isolates.³ Continuous...

Hybridoma Screening by Antigen Capture: Reverse Dot Blot

A dot blot is widely used to determine the productivity of a given hybridoma. This assay can also be used to screen a fusion or subclone plate for productive hybridoma clones. First, a nitrocellulose membrane is coated with an affinity-purified goat or rabbit anti-mouse immunoglobulin and then incubated with hybridoma tissue culture supernatant. Monoclonal antibodies in the supernatant are then “captured” on the coated nitrocellulose membrane surface and detected by screening with horseradish peroxidase (HRP).

The Translational Role of MUC8 in Salivary Glands: A Potential Biomarker for Salivary Stone Disease?

Mucin (MUC) 8 has been shown to play an important role in respiratory disease and inflammatory responses. In the present study, we investigated the question of whether MUC8 is also produced and secreted by salivary glands and whether it may also play a role in the oral cavity in the context of inflammatory processes or in the context of salivary stone formation. Tissue samples from parotid and submandibular glands of body donors (n = 6, age range 63–88 years), as well as surgically removed salivary stones from patients (n = 38, age range 48–72 years) with parotid and submandibular stone disease were immunohistochemically analyzed targeting MUC8 and TNFα. The presence of MUC8 in salivary stones was additionally analyzed by dot blot analyses. Moreover, saliva samples from patients (n = 10, age range 51–72 years), who had a salivary stone of the submandibular gland on one side were compared with saliva samples from the other “healthy” side, which did not have a salivary stone, by ELISA. Positive MUC8 was detectable in the inter- and intralobular excretory ducts of both glands (parotid and submandibular). The glandular acini showed no reactivity. TNFα revealed comparable reactivity to MUC8 in the glandular excretory ducts and also did not react in glandular acini. Salivary stones demonstrated a characteristic distribution pattern of MUC8 that differed between parotid and submandibular salivary stones. The mean MUC8 concentration was 71.06 ng/mL in female and 33.21 ng/mL in male subjects (p = 0.156). Saliva from the side with salivary calculi contained significantly (15-fold) higher MUC8 concentration levels than saliva from the healthy side (p = 0.0005). MUC8 concentration in salivary stones varied from 4.59 ng/mL to 202.83 ng/mL. In females, the MUC8 concentration in salivary stones was significantly (2.3-fold) higher, with an average of 82.84 ng/mL compared to 25.27 ng/mL in male patients (p = 0.034). MUC8 is secreted in the excretory duct system of salivary glands and released into saliva. Importantly, MUC8 salivary concentrations vary greatly between individuals. In addition, the MUC8 concentration is gender-dependent (♀️ > ♂). In the context of salivary stone diseases, MUC8 is highly secreted in saliva. The findings support a role for MUC8 in the context of inflammatory events and salivary stone formation. The findings allow conclusions on a gender-dependent component of MUC8.

Overexpression of chalcone isomerase A gene in Astragalus trigonus for stimulating apigenin

Apigenin is one of the most studied flavonoids and is widely distributed in the plant kingdom. Apigenin exerts important antioxidant, antibacterial, antifungal, antitumor activities, and anti-inflammatory effects in neurological or cardiovascular disease. Chalcone isomerase A (chiA) is an important enzyme of the flavonoid biosynthesis pathway. In order to enhance the apigenin production, the petunia chi A gene was transformed for Astragalus trigonus. Bialaphos survived plants were screened by PCR, dot blot hybridization and RT-PCR analysis. Also, jasmonic acid, salicylic acid, chitosan and yeast extract were tested to evaluate their capacity to work as elicitors for apigenin. Results showed that yeast extract was the best elicitor for induction of apigenin with an increase of 3.458 and 3.9 fold of the control for calli and cell suspension culture, respectively. Transformed cell suspension showed high apigenin content with a 20.17 fold increase compared to the control and 6.88 fold more than the yeast extract treatment. While, transformed T1 calli derived expressing chiA gene produced apigenin 4.2 fold more than the yeast extract treatment. It can be concluded that the highest accumulation of apigenin was obtained with chiA transgenic cell suspension system and it can be utilized to enhancement apigenin production in Astragalus trigonus.

The prevalence of human papillomavirus and bacterial vaginosis among young women in China: a cross-sectional study

Background The natural history of human papillomavirus (HPV) is influenced by vaginal microenvironment disorders, such as bacterial vaginosis (BV). The objective of this study was to assess the epidemiology of HPV combined with BV prevalence among Chinese women aged 20–35 years. Methods A total of 2000 sexually active women aged 20–35 years voluntarily enrolled in this study and underwent a ThinPrep cytologic test and PCR-reverse dot blot human papillomavirus genotyping (PCR-RDB HPV test). BV was diagnosed if clue cells were observed (20% more than epithelial cells). Results The overall HPV infection rate in this population was 16.2% (324/2000). Compared with HPV-negative individuals, BV prevalence was higher in the High-risk human papillomavirus (HR-HPV) (5.9% vs. 3.1%, P < 0.001). BV and HPV-51, -52 infection were more commonly associated with each other. In patients with cervical lesions (≥ CIN 1), the BV prevalence rate was higher than in patients with negative for intraepithelial lesion or malignancy (NILM) (11.9% vs. 3.8%, P = 0.002). Conclusion BV was found to be related to HPV-51, -52 infections and cervical lesions. To better manage HPV infected population, more attention should be paid to the prevention and proper treatment of BV.

Aptamers based sensing of pregnancy associated glycoproteins (PAG) of bovine for early pregnancy detection

Tosyl activated magnetic beads were used for aptamer selection against PAG- 7 and 18 proteins of bovine origin. PAG proteins were immobilized on beads with further addition of biotin tagged aptamer library. The recognition of aptamers with PAG was identified by ST-HRP based approach which was colorimetric in nature. The selected aptamers were sequenced and at the same time several new aptamers were identified. Later M-fold structure and G-quadruplex score of aptamers were analyzed for their selection. Those aptamers having high G value and complex structure were chosen. In dot blot assay, aptamers recognized PAG protein in an animal after 42 days of artificial insemination which later given birth to a healthy calf. Further the cross reactivity with serum of 0th day animal (post AI) or with non pregnant animal serum was minimal. Aptamers have also shown interaction with PAG protein of buffalo origin. These selected aptamers have commercial application especially in development of biosensors for early detection of pregnancy in bovine.