A recent update on the use of microbial transglutaminase for the generation of biotherapeutics.

The recent scientific progresses on the use of enzyme-mediated reactions in organic, non-aqueous and aqueous media have significantly supported the growing demand of new biotechnological and/or pharmacological products. Today, a plethora of microbial enzymes, used as biocatalysts, are available. Among these, microbial transglutaminases (MTGs) are broadly used for their ability to catalyse the formation of an isopeptide bond between the γ-amide group of glutamines and the ε-amino group of lysine. Due to their promiscuity towards primary amine-containing substrates and the more stringent specificity for glutamine-containing peptide sequences, several combined approaches can be tailored for different settings, making MTGs very attractive catalysts for generating protein-protein and protein small molecule's conjugates. The present review offers a recent update on the modifications attainable by MTG-catalysed bioreactions as reported between 2014 and 2019. In particular, we present a detailed and comparative overview on the MTG-based methods for proteins and antibodies engineering, with a particular outlook on the synthesis of homogeneous antibody-drug conjugates.

Automatic procedures for the synthesis of difficult peptides using oxyma as activating reagent: A comparative study on the use of bases and on different deprotection and agitation conditions.

Solid-Phase Peptide Synthesis (SPPS) is a rapid and efficient methodology for the chemical synthesis of peptides and small proteins. However, the assembly of peptide sequences classified as "difficult" poses severe synthetic problems in SPPS for the occurrence of extensive aggregation of growing peptide chains which often leads to synthesis failure. In this framework, we have investigated the impact of different synthetic procedures on the yield and final purity of three well-known "difficult peptides" prepared using oxyma as additive for the coupling steps. In particular, we have comparatively investigated the use of piperidine and morpholine/DBU as deprotection reagents, the addition of DIPEA, collidine and N-methylmorpholine as bases to the coupling reagent. Moreover, the effect of different agitation modalities during the acylation reactions has been investigated. Data obtained represent a step forward in optimizing strategies for the synthesis of "difficult peptides".

Ultra-performance liquid chromatography/multiple reaction monitoring mass spectrometry quantification of trastuzumab in human serum by selective monitoring of a specific peptide marker from the antibody complementarity-determining regions.

RATIONALE: Because of the large molecular weight, the structural complexity and the similarity with endogenous immunoglobulins present in high concentrations, in vivo quantitative studies with therapeutic monoclonal antibodies are particularly challenging. In this work, an UPLC/MRM MS-based methodology is described for the quantification of trastuzumab in human serum by monitoring a novel specific peptide marker located within its heavy chain Complementarity-Determining Region (CDR). METHODS: For maximum sensitivity and selectivity, specific transitions of this diagnostic proteotypic peptide were optimized and monitored at m/z 364.1 â†’ 437.3 (quantitation ion) and m/z 364.1 â†’ 358.0 (confirmation ion). As a proof-of-concept, the methodology was applied to the determination of trastuzumab in human serum over a clinically relevant range from 0.02 to 200 µg/mL. The methodology has been evaluated in terms of specificity, linearity, accuracy, precision, detection and quantitation limits. RESULTS: An excellent linear response has been obtained in the range from 0.036 to 3.6 fmol/µL for the standard peptide and from 0.03 to 285 fmol/µL for the trastuzumab in human serum with typical R2 values of 0.99. The limit of detection (LOD) and limit of quantification (LOQ) are 0.005 fmol/µL and 0.05 fmol/µL, respectively, with mean bias and RSD values of 18% and 1%, respectively, for quality control samples. CONCLUSIONS: The strategy used to set up the UPLC/MRM MS methodology based on monitoring specific peptide markers within CDRs can be potentially applied to the detection and quantification of other humanized or human mAbs in biological fluids. Copyright © 2017 John Wiley & Sons, Ltd.

The γ-parameter of anomalous diffusion quantified in human brain by MRI depends on local magnetic susceptibility differences.

Motivated by previous results obtained in vitro, we investigated the dependence of the anomalous diffusion (AD) MRI technique on local magnetic susceptibility differences (Δχ) driven by magnetic field inhomogeneity in human brains. The AD-imaging contrast investigated here is quantified by the stretched-exponential parameter γ, extracted from diffusion weighted (DW) data collected by varying diffusion gradient strengths. We performed T2* and DW experiments in eight healthy subjects at 3.0T. T2*-weighted images at different TEs=(10,20,35,55)ms and DW-EPI images with fourteen b-values from 0 to 5000s/mm2 were acquired. AD-metrics and Diffusion Tensor Imaging (DTI) parameters were compared and correlated to R2* and to Δχ values taken from literature for the gray (GM) and the white (WM) matter. Pearson's correlation test and Analysis of Variance with Bonferroni post-hoc test were used. Significant strong linear correlations were found between AD γ-metrics and R2* in both GM and WM of the human brain, but not between DTI-metrics and R2*. Depending on Δχ driven magnetic field inhomogeneity, the new contrast provided by AD-γ imaging reflects Δχ due to differences in myelin orientation and iron content within selected regions in the WM and GM, respectively. This feature of the AD-γ imaging due to the fact that γ is quantified by using MRI, may be an alternative strategy to investigate, at high magnetic fields, microstructural changes in myelin, and alterations due to iron accumulation. Possible clinical applications might be in the field of neurodegenerative diseases.

Mean diffusivity discriminates between prostate cancer with grade group 1&2 and grade groups equal to or greater than 3.

PURPOSE: To test the potential ability of mean diffusivity (MD) and fractional anisotropy (FA) in discriminating between PCa of grade group (GG) 1&2, and GGs≥3. MATERIAL AND METHODS: Diffusion Tensor Imaging (DTI) experiments at 3T in a cohort of 38 patients with PCa (fifty lesions in total) were performed, by using different diffusion weights (b values) up to 2500s/mm(2). Gleason score (GS) and GG data were correlated with DTI parameters (MD and FA) estimated in PCa. The relation between DTI measures and GS was tested by the linear correlation analysis (Pearson's coefficient). One-way analysis of variance to check the statistical significance of the difference between GG 1&2 and GGs 3, 4, 5, ≥3 was used. Results were reported for each of the three b-values ranges: 0-800s/mm(2), 0-1500s/mm(2), 0-2500s/mm(2). RESULTS: A negative correlation was found between MD and GS. The highest linear correlation was observed when the fit was performed with data acquired in the b-values range 0-2500s/mm(2). MD values were significantly different between GG 1&2 and GG=3 and between GG 1&2 and GG ≥3. Moreover this difference is better defined when high b values (higher than b=800s/mm(2)) are used. The specificity, sensitivity and accuracy in the discrimination between GG 1&2 and GG=3 were: 90%, 66.7% and 82.4%, respectively when MD was estimated in the b-values range 0-2500s/mm(2) while these values were 85%, 58.3% and 78.4% when MD was estimated in the b-values range 0-800s/mm(2). Conversely FA did not discriminate between GG 1&2 and GG ≥3, at any investigated b-values range. CONCLUSION: This study suggests that MD estimation in PCa, obtained from DTI acquired at high b-values, can contribute to the diagnosis and grading of prostate cancer while FA is not a useful parameter for this purpose.

Design, conformational studies and analysis of structure-function relationships of PTH (1-11) analogues: the essential role of Val in position 2.

The N-terminal 1-34 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it elicits all the biological responses characteristic of the native intact PTH. Recent studies reported potent helical analogues of the PTH (1-11) with helicity-enhancing substitutions. This work describes the synthesis, biological activity, and conformational studies of analogues obtained from the most active non-natural PTH (1-11) peptide H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH2; specifically, the replacement of Val in position 2 with D-Val, L-(αMe)-Val and N-isopropyl-Gly was studied. The synthesized analogues were characterized functionally by in-cell assays and their structures were determined by CD and NMR spectroscopy. To clarify the relationship between the structure and activity, the structural data were used to generate a pharmacophoric model, obtained overlapping all the analogues. This model underlines the fundamental functional role of the side chain of Val2 and, at the same time, reveals that the introduction of conformationally constrained Cα-tetrasubstituted α-amino acids in the peptides increases their helical content, but does not necessarily ensure significant biological activity.

Effect of inorganic and organic ligands on the sorption/desorption of arsenate on/from Al-Mg and Fe-Mg layered double hydroxides.

This paper describes the sorption of arsenate on Al-Mg and Fe-Mg layered double hydroxides as affected by pH and varying concentrations of inorganic and organic ligands, and the effect of residence time on the desorption of arsenate by ligands. The capacity of ligands to inhibit the fixation of arsenate followed the sequence: nitrate

Peptide-peptoid hybrids based on (1-11)-parathyroid hormone analogs.

A series of peptide-peptoid hybrids, containing N-substituted glycines, were synthesized based on the H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH(2) (Har = Homoarginine) as the parent parathyroid hormone (1-11) analog. The compounds were pharmacologically characterized in their agonistic activity at the parathyroid hormone 1 receptor.

[Endometrial ablation versus hysterectomy in women treated with tamoxifen]. / Ablazione endometriale versus isterectomia in pazienti in trattamento con tamoxifene.

AIM: The aim of our study is the assessment of the importance of the endometrial ablation versus hysterectomy in patients treated with tamoxifen for previous breast cancer. METHODS: Fifty-eight outpatients in therapy with tamoxifen for 1 year were controlled in the Department of Gynaecology of the University of Naples. We have selected these patients in two groups: group A, with 28 women with abnormal uterine bleeding and endometrial thickness >8 mm and group B, with 30 normal endometrium asymptomatic women. All patient of group A and 18 of group B were treated with endometrial ablation. RESULTS: Next follow-up showed normal hysteroscopy figures in 89% of cases and 5% of cases needed a hysterectomy for new abnormal uterine bleeding and cytology. CONCLUSION: Our results show the utility of endometrial ablation especially in selected cases in therapy with tamoxifen for previous breast cancer.

Locoregional IL-2 therapy in the treatment of colon cancer. Cell-induced lesions of a murine model.

BACKGROUND: Local therapy with IL-2 may be very effective in the treatment of different forms of cancer. The aim of this study was to determine the effectiveness of IL-2 locoregional application in the treatment of colon cancer. MATERIALS AND METHODS: Twenty eight syngenic BDIX rats were utilized in this study. The rats were divided into two groups of fourteen animals: group T (treatment) and group C (control). All rats of both groups were injected, under the splenic capsule, with T 10(7) DHD/K2/ TRb neoplastic cells. Then, within and around the site of the previous inoculation, the T group was injected with 1 ml of glucosate solutions + 0.1% albumin (BSA) containing 2.5 x 10(6) IU of IL-2 ( Proleukin-Chiron), whereas the C group was injected with 1 ml of BSA alone. After three weeks, rats were sacrificed and the liver and spleen were removed. The following parameters were considered: volume and weight, neoplastic-non neoplastic tissue index of the spleen, mitotic index and vascular density of splenic and hepatic lesions. RESULTS: All the studied parameters showed statistically significant differences in treated and untreated animals. CONCLUSION: This study of a murine model demonstrated that IL-2 locoregional therapy may be effective in the treatment of colon cancer.

Lymphadenectomy in gastric cancer: influence on prognosis of lymph node count.

To better understand the role of the number of lymph nodes retrieved on long-term outcome of gastric cancer treatment, 154 patients who had undergone curative resection, with dissection of >15 nodes were retrospectively studied. Dissection of perigastric and extraperigastric lymph nodes, defined as 'extended' (>26 nodes dissected) in 39 cases and 'limited' (< or = 26 nodes dissected) in 115 cases, was performed. A total of 3479 lymph nodes (mean 22.6 per specimen), were dissected and of these 721 showed metastases. A mean of 8.1 lymph node metastases, per metastatic case, was found. Regression analysis showed no independent factor associated with the extent of lymphadenectomy. Depth of wall invasion (p=0.000) and histological growth pattern (p=0.044) were independently associated with the number of lymph nodes involved (pN0, pN1 1-7, pN2 >7). The cumulative 5-year survival rate was 47% in patients without lymph node metastases; 29% in those with 1-7 nodes involved and 17% in those with >8 nodes involved (p=0.002). Receiver operating characteristic (ROC) curve analysis, in 65 nodenegative cancer cases, demonstrated an area under the curve for vital status (alive or dead) of 0.602 (95% CI: 0.473 - 0.721). All node-negative cases with a number equivalent to or exceeding the cutoff point of 23 nodes were alive. ROC analysis showed 11 to be the cutoff number of metastasized lymph nodes in correlation with vital status. Almost all those patients in whom the number of positive nodes was equivalent to, or exceeded the cutoff point had died (area under the ROC curve 0.633; 95% CI: 0.524 - 0.733). ROC analysis showed that the cutoff lymph node ratio, in relation to vital status, was 0.33. The majority of patients at or above this cutoff point had died (area under ROC curve 0.682; 95% CI: 0.574 - 0.776). Multivariate survival analysis showed that lymph node ratio was the only independent prognostic factor (p=0.001). The present findings suggest that, in lymphadenectomy with at least 15 nodes, the number and status of regional nodes dissected, irrespective of the location, provide reliable prognostic information on curatively resected gastric carcinomas.

Cancer precursor lesions in intact stomach Helicobacter pylori gastritis and in resected stomach gastritis.

Hemigastrectomy for benign disease and Helicobacter pylori infection are risk conditions for the development of gastric cancer. Aim of the study was to compare gastric histology and precursor lesions of malignancy in these two conditions. The hemigastrectomy group included 351 consecutively endoscoped subjects operated for gastroduodenal benign disease. Six to ten biopsy specimens were routinely taken from the residual gastric mucosa. The intact stomach group included 2097 consecutively endoscoped symptomatic subjects, who did not receive eradication therapy against H. pylori. The histological findings were classified as normal mucosa (NM), chronic non atrophic gastritis (CNAG), chronic atrophic gastritis (CAG), intestinal metaplasia (IM) and dysplasia (DYS). One thousand and three intact stomachs were H. pylori negative, and 1094 showed H. pylori colonization. The age over fifty was a significant risk factor for the occurrence of IM (OR 2.52, P < or = 0.001) and DYS (OR 3.46, P < or = 0.001), while Hp-positivity was a risk factor for CNAG (OR 1.81, P < or = 0.001) and CAG (OR 3.88, P < or = 0.001). Gastroresection was associated to higher risk for CNAG (OR 1.53, P < or = 0.001) and DYS (OR 4.31, P < or = 0.001) and to a lower risk of CAG (OR 0.49, P < or = 0.001). Both in males and females the risk for CNAG was significantly higher in Hp-positive (males OR 1.92, P=0.000; females OR 1.70, P=0.000) and gastrectomized subjects (males OR 2.06, P=0.000; females OR 2.43, P=0.000). Gastrectomized males, furthermore, showed an increased risk for DYS (OR 5.82, P=0.000). The aged Hp-negative and Hp-positive subjects evidenced a significant risk for IM (respectively OR's 3.42, P=0.000 and 4.85, P=0.000); the risk for DYS was significant in aged Hp-negative subjects (OR 4.09 P < or = 0.020). The Hp-positive individuals evidenced a significant risk for metaplastic mucosal changes (OR 38.17, P=0.000). Subjects aged over forty at the time of surgery and those with a longer postoperative follow up endoscopy presented an increased risk for CNAG of the residual mucosa (respectively OR's 2.75, P=0.000 and 5.25, P=0.000). CNAG and IM were the most frequently observed mucosal lesions both in subjects operated for duodenal and gastric ulcer (respectively OR's 4.02, P=0.000 and 3.00, P=0.000). Our data support that hemigastrectomy for benign disease and H. pylori infection may induce an increased incidence for histological precursor lesions for gastric malignancy and suggest that carcinogenesis in a resected stomach may be different from that in the intact stomach.

Favorable toxicity profile of raltitrexed in elderly patients treated for colorectal cancer: a case series.

BACKGROUND: Age-related physiological changes may lead to an increased toxicity of chemotherapy in the elderly, thus making tumor treatment difficult in this increasing subset of patients. OBJECTIVE: Since many trials claimed a favorable therapeutic index with raltitrexed, the aim of our preliminary study was to evaluate the anticancer activity and the toxic profile of this drug in the elderly. METHODS: Thirteen elderly patients with colorectal cancer, aged 75-90 years, were enrolled in a monochemotherapy treatment with raltitrexed. Due to their advanced age, the drug was administered with a 33% reduction of the dose. RESULTS: One partial response, four disease stabilizations, and two disease progressions were observed in 7 patients with advanced colorectal cancer. The patients with response or disease stabilisation had a satisfactory time to progression. Four out of 6 patients treated in the adjuvant setting for Dukes' C colorectal cancer remain disease free at observation periods of 15+ to 29+ months. Toxicity was virtually absent in all patients. CONCLUSIONS: The activity of monochemotherapy with raltitrexed appears to be appealing, above all because it is observed in the absence of toxicity. Though recent reports suggest some concern about severe complications of treatment with raltitrexed, administration of reduced doses of this drug seems to be a putative therapy for those patients who, because of their age, are highly susceptible to the adverse effects of chemotherapy.

Maximum gastric cancer diameter as a prognostic indicator: univariate and multivariate analysis.

It was suggested that there are no stronger prognostic factors in gastric cancer than nodal involvement or depth of wall invasion. The present paper evaluated the influence of maximum tumor diameter (MTD) value, measured on fixed resected specimens, on the extent of disease progression and the outcome in gastric cancer patients. Clinicopathological data were retrospectively retrieved from records of 122 patients who underwent curative gastrectomy. The patients' MTD values were grouped as follows: MTD1, up to 26 mm; MTD2, between 26 and 50 mm; and MTD3, over 50 mm. The three groups evidenced significant differences with regard to 5 year survival (MTD1: 54%, MTD2: 31%, MTD3: 20%; p = 0.00027), furthermore they were significantly different with respect to the type of gastrectomy (p = 0.021), depth wall invasion (p = 0.000), lymphatic microinvasion (p = 0.014), perineural microinvasion (p = 0.017), stromal reaction (p = 0.025), and stage (p = 0.035). ROC curve analysis individuated a best accurate MTD threshold value for nodal involvement of 32 mm (sensitivity = 56.6%; specificity = 60.9%; positive predictive value = 52.6%; negative predictive value = 64.6%). The logistic regression analysis suggested that the depth of wall invasion was the only independent variable associated with MTD value (p = 0.0005). Multivariate analysis showed that independent prognostic risk factors were sex (p < 0.0025), number of involved nodes (p < 0.001) and MTD (p < 0.001). In conclusion, the maximum tumor diameter value of gastric cancer may be a factor with greater prognostic implications than previously believed.

Intracranial hemorrage due to brain metastases in an Italian HCV patient with hepatocellular carcinoma.

Brain metastases from hepatocarcinoma are exceptional and only a few cases have been reported in the literature, mainly from Far-Eastern countries. Clinical diagnosis in asymptomatic patients with preserved liver function is difficult and usually late. In some cases, cerebral metastasis is the initial manifestation of HCC and patients may develop intracerebral hemorrage and have a stroke-like presentation. We report on the first Italian case of cerebral metastases from multifocal hepatocellular carcinoma in an asymptomatic HbsAg negative patient with unknown HCV related chronic hepatitis and no evidence of liver cirrhosis. For many years he had a mild liver enzyme elevation and the presence of multiple misinterpreted hypoechogenic hepatic lesions. The hepatic tumor spread to the lungs and the brain and the patient developed two major episodes of intracranial hemorrage. He had two nodular lesions in the brain and alpha-fetoprotein levels were more than 10,000 ng/ml. He died from neurologic causes, without major signs of liver failure.

Presacral myelolipoma in a patient with colon carcinoma.

Myelolipoma is an uncommon benign tumor, composed of an admixture of adipose tissue and hematopoietic cells, usually discovered within the adrenal glands. Extra-adrenal myelolipomas are rare and approximately half of the reported cases were presacral. We report a case of an asymptomatic presacral myelolipoma measuring 9x8x7 cm, incidentally found in a 71-year-old woman with a stenosing colon cancer. Ultrasonography, computed tomography and magnetic resonance imaging of the presacral mass evidenced a well circumscribed tumor with heterogeneous features due to a nonuniform composition. Surgical excision of the mass was performed.