Circulating tumor DNA in neoadjuvant-treated breast cancer reflects response and survival.

BACKGROUND: Pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) is strongly associated with favorable outcome. We examined the utility of serial circulating tumor DNA (ctDNA) testing for predicting pCR and risk of metastatic recurrence. PATIENTS AND METHODS: Cell-free DNA (cfDNA) was isolated from 291 plasma samples of 84 high-risk early breast cancer patients treated in the neoadjuvant I-SPY 2 TRIAL with standard NAC alone or combined with MK-2206 (AKT inhibitor) treatment. Blood was collected at pretreatment (T0), 3 weeks after initiation of paclitaxel (T1), between paclitaxel and anthracycline regimens (T2), or prior to surgery (T3). A personalized ctDNA test was designed to detect up to 16 patient-specific mutations (from whole-exome sequencing of pretreatment tumor) in cfDNA by ultra-deep sequencing. The median follow-up time for survival analysis was 4.8 years. RESULTS: At T0, 61 of 84 (73%) patients were ctDNA positive, which decreased over time (T1: 35%; T2: 14%; and T3: 9%). Patients who remained ctDNA positive at T1 were significantly more likely to have residual disease after NAC (83% non-pCR) compared with those who cleared ctDNA (52% non-pCR; odds ratio 4.33, P = 0.012). After NAC, all patients who achieved pCR were ctDNA negative (n = 17, 100%). For those who did not achieve pCR (n = 43), ctDNA-positive patients (14%) had a significantly increased risk of metastatic recurrence [hazard ratio (HR) 10.4; 95% confidence interval (CI) 2.3-46.6]; interestingly, patients who did not achieve pCR but were ctDNA negative (86%) had excellent outcome, similar to those who achieved pCR (HR 1.4; 95% CI 0.15-13.5). CONCLUSIONS: Lack of ctDNA clearance was a significant predictor of poor response and metastatic recurrence, while clearance was associated with improved survival even in patients who did not achieve pCR. Personalized monitoring of ctDNA during NAC of high-risk early breast cancer may aid in real-time assessment of treatment response and help fine-tune pCR as a surrogate endpoint of survival.

Volatile organic compound measurements point to fog-induced biomass burning feedback to air quality in the megacity of Delhi.

We report the first ambient measurements of thirteen VOCs for investigations of emissions and air quality during fog and non-fog wintertime conditions at a tower site (28.57° N, 77.11° E, 220 m amsl) in the megacity of Delhi. Measurements of acetonitrile (biomass burning (BB) tracer), isoprene (biogenic emission tracer in daytime), toluene (a traffic exhaust tracer) and benzene (emitted from BB and traffic), together with soluble and reactive oxygenated VOCs such as methanol, acetone and acetaldehyde were performed during the winters of 2015-16 and 2016-17, using proton transfer reaction mass spectrometry. Remarkably, ambient VOC composition changes during fog were not governed by solubility. Acetaldehyde, toluene, sum of C8-aromatics (e.g. xylenes), sum of C9-aromatics (e.g. trimethyl benzenes) decreased by ≥30% (>95% confidence interval), whereas acetonitrile and benzene showed significant increases by 20% (>70% confidence interval), even after accounting for boundary layer dilution. During fog, the lower temperatures appeared to induce an emissions feedback from enhanced open BB within Delhi for warming, releasing both gaseous and aerosol pollutants with consequences for fog chemistry, sustenance and intensity. The potential feedback is important to consider for improving current emission parametrizations in models used for predicting air quality and fog in such atmospheric environments.

Novel Curcumin Diclofenac Conjugate Enhanced Curcumin Bioavailability and Efficacy in Streptococcal Cell Wall-induced Arthritis.

Curcumin-diclofenac conjugate as been synthesized by esterification of phenolic group of curcumin with the acid moiety of diclofenac, and characterized by mass spectrometry, NMR, FTIR, DSC, thermogravimetric analysis and X-ray diffraction analysis. The relative solubility of curcumin-diclofenac conjugate, curcumin and diclofenac; stability of curcumin-diclofenac conjugate in intestinal extract; permeability study of curcumin-diclofenac conjugate using the everted rat intestinal sac method; stability of curcumin-diclofenac conjugate in gastrointestinal fluids and in vitro efficacy have been evaluated. In vivo bioavailability of curcumin-diclofenac conjugate and curcumin in Sprague-Dawley rats, and antiarthritic activity of curcumin-diclofenac conjugate, curcumin and diclofenac in modified streptococcal cell wall-induced arthritis model in Balb/c mice to mimic rheumatoid arthritis in humans have also been studied. In all of the above studies, curcumin-diclofenac conjugate exhibited enhanced stability as compared to curcumin; its activity was twice that of diclofenac in inhibiting thermal protein denaturation taken as a measure of in vitro antiinflammatory activity; it enhanced the bioavailability of curcumin by more than five folds, and significantly (P<0.01) alleviated the symptoms of arthritis in streptococcal cell wall-induced arthritis model as compared to both diclofenac and curcumin.

Over-expression of gene encoding heat shock protein 70 from Mycobacterium tuberculosis and its evaluation as vaccine adjuvant.

BACKGROUND: Heat shock proteins (Hsps) are evolutionary ancient and highly conserved molecular chaperons found in prokaryotes as well as eukaryotes. Hsp70 is a predominant member of Hsp family. Microbial Hsp70s (mHsp70s) have acquired special significance in immunity since they have been shown to be potent activators of the innate immune system and generate specific immune responses against tumours and infectious agents. OBJECTIVES: The present study was aimed to clone express and purify recombinant Hsp70 from the Mycobacterium tuberculosis and characterise it immunologically. The study also aimed at determining the potential of recombinant M. tuberculosis heat shock protein (rMTB-Hsp70) as adjuvant or antigen carrier. MATERIALS AND METHODS: Cloning of M. tuberculosis heat shock protein (MTB-Hsp70) amplicon was carried out using the pGEMT-Easy vector although for expression, pProExHTb prokaryotic expression vector was used. Purification of recombinant Hsp70 was carried out by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. For immunological characterization and determining the adjuvant effect of MTB-Hsp70, BALB/c mice were used. The data obtained was statistically analysed. RESULTS: Hsp70 gene was cloned, sequenced and the sequence data were submitted to National Center for Biotechnology Information (NCBI). Recombinant MTB-Hsp70 was successfully over-expressed using the prokaryotic expression system and purified to homogeneity. The protein was found to be immunodominant. Significant adjuvant effect was produced by the rMTB-Hsp70 when inoculated with recombinant outer membrane protein 31; however, effect was less than the conventionally used the Freund's adjuvant. CONCLUSION: Protocol standardised can be followed for bulk production of rHsp70 in a cost-effective manner. Significant adjuvant effect was produced by rMTB-Hsp70; however, the effect was than Freund's adjuvant. Further, studies need to be carried out to explore its applicability as carrier of antigen.

Preparation, optimisation and characterisation of novel wound healing film dressings loaded with streptomycin and diclofenac.

Streptomycin (STP) and diclofenac (DLF) loaded film dressings were prepared by blending Polyox(®) (POL) with four hydrophilic polymers [hydroxypropylmethylcellulose (HPMC), carrageenan (CAR), sodium alginate (SA) or chitosan (CS)] using glycerol (GLY) as plasticiser. The films were characterised by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy, texture analysis (tensile and swelling characteristics) and in vitro dissolution profiles using Franz diffusion cell. SEM showed homogeneous morphology for both blank (BLK) and drug loaded (DL) films. Films prepared by blending of POL with the other polymers showed a reduction in the crystallisation of POL in descending order of SA>CS>HPMC>CAR respectively. DSC and XRD showed no crystalline peaks of STP and DLF suggesting molecular dispersion of both drugs as well as possible drug interaction with negatively charged sulphate ions present in CAR. The DL films did not show any IR bands of both drugs, confirming the DSC and XRD results. POL-CAR-BLK films showed higher tensile strength (12.32±1.40 MPa) than the POL-CAR-DL films (9.52±1.12 MPa). DL films plasticised with 25%w/w GLY revealed soft and tough (tensile strength 1.02±0.28 MPa, % elongation 1031.33±16.23) formulations. The swelling capacities of POL-CAR-BLK and POL-CAR-DL films were (733.17±25.78%) and (646.39±40.39%), increasing to (1072.71±80.30%) and (1051±86.68%) for POL-CAR-BLK-25% GLY and POL-CAR-DL-25% GLY respectively. POL-CAR-DL films showed significantly (n=3, p<0.0318) lower cumulative release of STP and DLF (52.11±1.34, 55.26±2.25) compared to POL-CAR-DL-25% GLY films (60.07±1.56, 63.39±1.92) respectively.

Comparative evaluation of real time PCR assay with conventional parasitological techniques for diagnosis of Trypanosoma evansi in cattle and buffaloes.

For comparative evaluation, a real time PCR assay was standardized by using TaqMan primer and probe targeting the internal transcribed spacer 1 (ITS-1) region of rRNA for Trypanosoma evansi and sensitivity was evaluated by using DNA, extracted from diethyleamino ethane cellulose purified trypanosomes and trypanosomes infected whole blood of mice. The minimum detection limit for purified trypanosomal DNA was 0.01 ng (≈ 0.33 genomic DNA of T. evansi) whereas for whole blood the minimum detection limit was 0.1 ng (≈ 6.12 genomic DNA). T. evansi infected mice blood samples were collected at different interval post infection and were analysed by conventional parasitological methods (CPT) viz. wet blood smear, thin blood smear, thick blood smear, quantitative buffy coat and real time PCR and found that TaqMan assay was two fold sensitive than CPT in case of in vivo infectivity in mice and gave positive signal at 36 h post infection where as QBC and blood smear examination was able to detect at 60 h and 72 h post infection respectively. A total 109 (80 cattle and 29 buffaloes) blood samples were collected from in and around Ludhiana district and analysed by CPT and real time PCR. The overall prevalence of T. evansi by CPT in cattle and buffaloes was 2.75 per cent. The prevalence rate was 2.5 per cent in cattle and 3.45 per cent in buffaloes. By real time PCR overall prevalence was 12.84 per cent in cattle and buffaloes, with a prevalence rate of 12.50 per cent in cattle and 13.79 per cent in buffaloes.

X-linked juvenile retinoschisis: localization between (DXS1195, DXS418) and AFM291wf5 on a single YAC.

We studied 17 pedigrees with 108 affected males with X-linked juvenile retinoschisis (RS; McKusick No. 31270) and have analyzed all of the known polymorphic markers in the RS region of Xp22.1-p22.2 between DXS987 and DXS41. By haplotype analyses we found 7 individuals who showed crossovers in this interval surrounding RS. We previously reported AFM291wf5 as the centromeric boundary, and this remains unchanged in the present study. A new recombination was identified on the telomeric side at (DXS1195, DXS418). Our data support the locus order Xpter--(DXS987, DXS207, DXS1053, DXS43)--(DXS1195, DXS418)--(RS, DXS257, DXS999)--(AFM291wf5, DXS443)--DXS1052--(DXS1226, DXS274, DXS41)--Xcen; loci grouped in parentheses could not be mutually ordered by our genetic data. Physical mapping has indicated a distance of at most 900-1,000 kb between (DXS1195, DXS418) and AFM291wf5. No recombination was observed between RS and DXS257 which lies in our new interval of interest, but one critical individual was not informative with this marker. Our data now define the smallest RS inclusion interval. This interval is contained on a single YAC from which we have identified expressed sequences as candidate genes for RS.

Linkage study of Best's vitelliform macular dystrophy (VMD2) in a large North American family.

Best's vitelliform macular dystrophy (VMD2) is an autosomal dominant retinal dystrophy for which the underlying biochemical cause is unknown. We used 11 genetic markers in the vicinity of the VMD2 gene in our study of a large North American family in which macular dystrophy characteristics overlap the broad definition of Best's disease. Significant evidence for linkage was found for markers D11S956 (Z = 5.88, theta = 0.04) and FCER1B (Z = 4.31, theta = 0.00). Recombination events localized the disease gene to the 5-cM interval D11S956-UGB, a genetic inclusion interval that substantially overlaps the VMD2 inclusion interval defined by recombinants at FCER1B and UGB observed by other research groups. The resulting exclusion of ROM1 from the genetic inclusion interval eliminates ROM1 defects as a possible cause of the disease in this family. Linkage studies of many families, including those that share most but not all features with classical Best's disease, will be needed to properly evaluate genetic heterogeneity and the range of phenotypic variation that can result from VMD2 defects.

Sorsby's fundus dystrophy in a family with a Ser-181-CVS mutation in the TIMP-3 gene: poor outcome after laser photocoagulation.

PURPOSE: Mutations in the TIMP-3 (tissue inhibitor of metalloproteinase-3) gene can cause Sorsby's fundus dystrophy (SFD) and lead to choroidal neovascular membrane (CNV) formation. We studied a large American family of Irish Protestant descent with CNV inherited as an autosomal dominant trait, to determine the phenoptype and to learn whether a mutation was present in TIMP-3. METHODS: Twelve members of 5 generations were evaluated clinically, with psychophysical and electroretinographic testing, and by fluorescein angiography. Blood samples for DNA extraction were obtained from 21 affected and unaffected family members and from 1 unrelated spouse. DNA sequence was determined, and affected individuals showed a Ser-181-Cys mutation in TIMP-3 exon 5. RESULTS: Observable pathology involved primarily the macula, and both the full-field ERG and visual fields were normal. Acute CNV occurred during the third through fifth decades, with second eyes typically also affected during the subsequent year. Three affected members complained of nyctalopia prior to developing CNV. A Ser-181-Cys mutation in the TIMP-3 gene cosegregated with CNV in 10 affected subjects but was absent in 3 relatives at risk and sufficiently old to trust the clinical designation of normalcy. Nine eyes of 6 family members were treated by laser photocoagulation by 5 different ophthalmologists for foveal and juxtafoveal CNV. All eyes had recurrent CNV and lost acuity to 20/300 or less within several months. CONCLUSIONS: Laser photocoagulation of CNV did not stem vision loss in this SFD family. Although possible benefits of laser treatment were not put to formal clinical trial owing to the limited number of Sorsby's cases, it appears that photocoagulation is not of long-term benefit for preserving vision loss from the TIMP-3 Ser-181-Cys mutation. Several younger family members with the mutation are thus far not clinically affected and are being followed up.

Refined genetic mapping of juvenile X-linked retinoschisis.

Juvenile X-linked retinoschisis (RS) is an eye disease that causes acuity reduction and peripheral visual field loss typically beginning early in life. In further work towards positional cloning of the RS gene, we restudied our previously reported seven large American families and one additional new family, with a total of 63 affected males. RS linkage analysis using microsatellite repeat markers gave the following results: DXS207 (Z = 24.89, theta = 0.01), DXS987 (Z = 24.04, theta 0.01) and DXS999 (Z = 14.70, theta = 0.00). Recombination events in four individuals were studied further with additional markers (AFM291wf5, DXS443, DXS1052, DXS274 and DXS1226), and a flanking interval was obtained (DXS43, DXS207, DXS987)-RS-(AFM291wf5, DXS443). This study moves the RS centromeric boundary to (AFM291wf5, DXS443), about 5.5 cM closer than the previously reported boundary at DXS274 and narrows the RS inclusion interval to about 3.7 cM (using distances from CEPH family data).

Immobilization of D-xylose (D-glucose) isomerase from a Chainia species.

D-Xylose isomerase is a heat-stable enzyme which isomerizes D-xylose into D-xylulose. D-Xylose isomerase from various species also isomerizes D-glucose into D-fructose. This enzyme is used in industry for the production of high-fructose corn syrup. The enzyme is specific for both, xylose and glucose. In most species xylose isomerase is localized intracellularly. However, in a rare actinomycete, Chainia sp. (NCL 82-5-1), xylose isomerase is present in both intracellular and extracellular compartments. We have previously purified and characterized intracellular enzyme from Chainia sp. In the present paper, we describe a procedure for immobilization of intracellular xylose isomerase on INDION 48-R by ionic binding. This method is inexpensive, does not require cross-linking agents and results in firm binding of the enzyme with the resin. The properties of immobilized enzyme such as pH optimum, substrate specificity, Km and inhibition by various metabolites are described and compared with those of purified, nonimmobilized enzyme.

Localization of the gene for pigment epithelium-derived factor (PEDF) to chromosome 17p13.1 and expression in cultured human retinoblastoma cells.

The gene for pigment epithelium-derived factor (PEDF) was localized to chromosome 17 by the analysis of three independent somatic cell hybrid panels. Fluorescence in situ hybridization shows a specific hybridization signal at the terminal portion of the short arm of chromosome 17. PCR analysis of somatic cell hybrids containing specific regions of 17 was subsequently used to sublocalize PEDF to 17p13.1-pter. PEDF thus maps to a region containing a number of cancer-related loci and thus must be considered a candidate gene for these cancers. Preliminary studies with cultured human Y79 retinoblastoma cells indicate that expression of PEDF is associated with relatively undifferentiated, proliferating cells rather than their differentiated, slow-growing counterparts. This and the fact that the PEDF protein can act as a potent neurotrophic differentiating agent suggest that PEDF is linked to proliferative events that terminate in final phenotypic determination within specific cell lineages.

Quantitative morphology of the testicular tubular epithelium in the water buffalo (Bubalus bubalis).

Ultrastructural features and morphometric evaluations of water buffalo seminiferous epithelium are reported for the 6 phases of the spermatogenic cycle. The relative Sertoli cell volume varies between 30% (phase 4) and 39% (phase 8), the calculated volume of a Sertoli cell between 7118 microns3 and 8968 microns3 (phase 4). Smooth ER is the organelle that exhibits the most prominent changes in Sertoli cells during the spermatogenic cycle: it occupies about 6% in phase 3 and 21% in phase 4. All spermatogenic cells of the same clone present cytoplasmic bridges among them. From preleptotene (about 470 microns3) to late diplotene (about 2300 microns3) the volume of a primary spermatocyte increases nearly 5-fold; their nuclear volumes increase 3.5-fold in the same period. Secondary spermatocytes are found only in phase 4 of the cycle. Due to partial cell necrosis and autolysis late maturation phase spermatids display not more than 25% of the size of early cap phase spermatids. 63% of all numerically possible germ cells disappear from the seminiferous epithelium during spermatogenesis. Particularly heavy cell loss is observed in phase 4 and involves the spermatogonial fraction as well as cells during the second meiotic division.

A conserved retina-specific gene encodes a basic motif/leucine zipper domain.

Using subtraction cloning, we have isolated a human cDNA, AS321, which is expressed in retina and retinoblastoma cell lines but not in any other tissue or cell line tested. AS321 mRNA is detected in all cells of neural retina, with a high level of expression in photoreceptors. The polypeptide sequence deduced from the cDNA reveals consensus phosphorylation sites for protein kinase A and proline-directed protein kinase. Its C-terminal region contains a basic motif and a leucine zipper domain similar to the DNA binding proteins of the jun and fos oncoprotein family, and it shows a strong similarity to the product of an avian retroviral oncogene, v-maf. The gene for AS321 is conserved during evolution and is expressed in vertebrate retina. We propose to name the gene NRL (neural retina leucine zipper.

Quantitative aspects of water buffalo (Bubalus bubalis) spermatogenesis.

In the buffalo, seminiferous tubules occupy about 82% of the testis. Spermatogenesis can be divided into 6 stages according to characteristic cellular associations in the seminiferous epithelium. A-spermatogonia have a volume of approximately 1,400 microns3 and the highest absolute mitochondrial volume of all spermatogenic cells. B-spermatogonia display cellular, nuclear and mitochondrial volumes of approximately half the values of A-spermatogonia. From preleptotene (approximately 470 microns3) to late diplotene (approximately 2,300 microns3), the volume* of primary spermatocytes increases nearly five-fold; their nuclear volumes increase by 3.5 times within the same period. During zygotene mitochondrial cristae start to dilate. Grouping of mitochondria by a dense intermitochondrial substance is most prominent during pachytene and diplotene. In pachytene the absolute size of the Golgi apparatus more than doubles, indicating a high secretory activity. Through zygotene only rER is encountered; in pachytene and diplotene a tubular sER makes its first appearance. Secondary spermatocytes are found only in stage 4 of the cycle. Due to partial cell necrosis and autolytic events, late maturation phase spermatids display no more than 25% of the size of cap phase spermatids. There is no morphological evidence for an active uptake and digestion of residual bodies by the Sertoli cells. Also, no lipid cycle is present in the buffalo seminiferous epithelium. Morphometric evaluations reveal that 63% of all theoretically possible germ cells disappear from the seminiferous epithelium during spermatogenesis. Heavy cell loss is observed in stage 4 of the cycle in the spermatogonial fraction as well as during the second meiotic division.

Serum prostate-specific antigen: hourly change/24 hours compared with prostatic acid phosphatase.

Simultaneous hourly changes in serum PSA and PAP levels per twenty-four-hour period was studied in 19 urologic patients with no prostatic disease. Serum PSA was determined using Hybritech PSA-R Kits, and PAP was determined using EIA method. PSA and PAP serum levels varied independently from each other. PSA level showed no diurnal variations. The level remained stable with minor variations around a low mean of 0.907 ng/mL, SD 0.09, and CV 9.9% in 16 of 19 (84%) patients, while PAP showed changes consistent with diurnal variations in 5 of those patients. Random variations with no discernible pattern in 7 and remained more or less constant in 4 other patients. Marked random variations in PSA serum level occurred in only 3 patients in the older age group and were accompanied by diurnal variations in PAP level in 2 patients and a constant high level of PAP in 1 patient. The highest PSA level obtained was 6.9 ng/mL which is important when selecting the cut-off level. PSA did not increase appreciably after prostatic massage, and on follow-up PSA returned to premassage level after twenty-four hours (except in 1 patient).

Streptomyces glucose/xylose isomerase has a single active site for glucose and xylose.

A kinetic method which allows one to evaluate whether an enzyme acting on two different substrates has one or two active sites was employed to study the active site of glucose isomerase which catalyses the isomerization of both glucose and xylose. The experimental data on the rates of hydrolysis of mixtures of various concentrations of glucose and xylose by the glucose isomerase from Streptomyces coincides well with the theoretical values calculated for the case of a single active site.

Purification and characterisation of glucose (xylose) isomerase from Chainia sp. (NCL 82-5-1).

Glucose (xylose) isomerase is an important enzyme in high fructose syrup industry. The enzyme generally occurs intracellularly and is specific for both glucose and xylose. A rare actinomycete Chainia sp. (NCL 82-5-1) produces extracellular specific glucose and xylose isomerases and an intracellular glucose (xylose) isomerase. The intracellular enzyme is isolated by cell autolysis and purified by preparative polyacrylamide gel electrophoresis. Its properties are studied and compared with those of extracellular specific xylose isomerase. The intracellular enzyme has a molecular weight of 1,58,000 daltons with four equal subunits of 40,700 daltons. The N-terminal amino acid sequence analysis shows Arg at the N-terminal. Diethylpyrocarbonate inhibited the enzyme and the inhibition kinetics study shows the presence of at least 2 essential His residues. The amino acid analysis shows the absence of Cys and a high proportion of hydrophobic and acidic amino acids.