L-asparaginase (ASNase) has played a key role in the management of acute lymphoblastic leukaemia (ALL). As an amidohydrolase, it catalyzes the hydrolysis of L-asparagine, a crucial step in the treatment of ALL. Various ASNase variants have evolved from diverse sources since it was first used in paediatric patients in the 1960s. This review describes the available ASNase and approaches being used to develop ASNase as a biobetter candidate.
The review discusses the Glycosylation and PEGylation techniques, which are frequently used to develop biobetter versions of the majority of the therapeutic proteins. Further, it explores current ASNase biobetters in therapeutic use and discusses the protein engineering and chemical modification approaches that were employed to reduce immunogenicity, extend protein half-life, and enhance protease stability of ASNase. Emerging strategies like immobilization and encapsulation are also highlighted as potential pathways for improving ASNase properties.
The purpose of the development of ASNase biobetter is to achieve a novel therapeutic candidate that could improve catalytic efficiency, in vivo stability with minimum glutaminase (GLNase) activity and toxicity. Modification of ASNase by immobilization and encapsulation or by fusion technologies like Albumin fusion, Fc fusion, ELP fusion, XTEN fusion, etc. can be exploited to develop a novel biobetter candidate suitable for therapeutic approaches.
This review emphasizes the importance of biobetter development for therapeutic proteins like ASNase. Improved ASNase molecules have the potential to significantly advance the treatment of ALL and have broader implications in the pharmaceutical industry.

Older adults with type 2 diabetes mellitus have an increased risk of fracture despite a paradoxically higher average bone mineral density. This study identified additional markers of fracture risk in this at-risk population. Non-esterified fatty acids and the amino acids glutamine/glutamate and asparagine/aspartate were associated with incident fractures.
OBJECTIVE: Type 2 diabetes mellitus (T2D) is associated with an increased risk of fracture despite a paradoxically higher bone mineral density. Additional markers of fracture risk are needed to identify at-risk individuals.
METHODS: The MURDOCK study is an ongoing study, initiated in 2007, of residents in central North Carolina. At enrollment, participants completed health questionnaires and provided biospecimen samples. In this nested case-control analysis, incident fractures among adults with T2D, age ≥ 50 years, were identified by self-report and electronic medical record query. Fracture cases were matched 1:2 by age, gender, race/ethnicity, and BMI to those without incident fracture. Stored sera were analyzed for conventional metabolites and targeted metabolomics (amino acids and acylcarnitines). The association between incident fracture and metabolic profile was assessed using conditional logistic regression, controlled for multiple confounders including tobacco and alcohol use, medical comorbidities, and medications.
RESULTS: 107 incident fractures were identified with 210 matched controls. Targeted metabolomics analysis included 2 amino acid factors, consisting of: 1) the branched chain amino acids, phenylalanine and tyrosine; and 2) glutamine/glutamate, asparagine/aspartate, arginine, and serine [E/QD/NRS]. After controlling for multiple risk factors, E/QD/NRS was significantly associated with incident fracture (OR 2.50, 95% CI: 1.36-4.63). Non-esterified fatty acids were associated with lower odds of fracture (OR 0.17, 95% CI: 0.03-0.87). There were no associations with fracture among other conventional metabolites, acylcarnitine factors, nor the other amino acid factors.
CONCLUSIONS: Our results indicate novel biomarkers, and suggest potential mechanisms, of fracture risk among older adults with T2D.

Circulating branched-chain amino acids (BCAAs, isoleucine, leucine, valine) and aromatic amino acids (AAAs, tyrosine and phenylalanine) predicted type 2 diabetes mellitus (T2DM) risk in a Caucasian population. Here, we assessed amino acid levels in relation to incident prediabetes among initially normoglycemic African Americans (AA) and European Americans (EA).
Using a nested case-control design, we studied 70 adults (35 AA, 35 EA) who developed prediabetes (progressors) and 70 matched participants who maintained normoglycemia (nonprogressors) during 5.5 years of follow-up in the Pathobiology of Prediabetes in a Biracial Cohort study. Assessments included plasma amino acid levels, insulin sensitivity, and beta-cell function.
The total level of all 18 amino acid assayed was significantly associated with lean mass (r = 0.36, P < 0.0001), waist circumference (r = 0.27, P = 0.001), fasting plasma glucose (r = 0.24, P = 0.005), HOMA-IR (r = 0.22, P = 0.01) and HDL cholesterol (r = -0.18, P = 0.03). Individual amino acid levels were significantly associated with insulin sensitivity and insulin secretion. Compared with nonprogressors, progressors had higher baseline levels of asparagine and aspartic acid (P <0.0001), glutamine/glutamic acid (P = 0.005) and phenylalanine (P = 0.02), and lower histidine (P = 0.02) levels. In fully-adjusted logistic regression models, aspartic acid/asparagine (OR 2.72 [95% CI 1.91-3.87]) and histidine (OR 0.90 [95% CI 0.85-0.96]) were the only amino acids that significantly predicted incident prediabetes.
Baseline plasma aspartic acid and asparagine levels predicted progression to prediabetes, whereas histidine levels were protective of prediabetes risk. Thus, the amino acid signature associated with prediabetes in a diverse population may be distinct from that previously linked to T2DM in Caucasians.

The amantadine-resistant S31N mutant of the influenza A M2 proton channel has become prevalent in currently circulating viruses. Here, we have solved an X-ray crystal structure of M2(22-46) S31N that contains two distinct conformational states within its asymmetric unit. This structure reveals the mechanism of adamantane resistance in both conformational states of the M2 channel. In the Inwardopen conformation, the mutant Asn31 side chain faces the channel pore and sterically blocks the adamantane binding site. In the Inwardclosed conformation, Asn31 forms hydrogen bonds with carbonyls at the monomer-monomer interface, which twists the monomer helices and constricts the channel pore at the drug binding site. We also examine M2(19-49) WT and S31N using solution NMR spectroscopy and show that distribution of the two conformational states is dependent on both detergent choice and experimental pH.

Discovery of a high-risk group for pancreatic cancer is important for prevention of pancreatic cancer. The present study was conducted as a nested case-control study including 170 pancreatic cancer cases and 340 matched controls of our population-based cohort study involving 30 239 subjects who answered a baseline questionnaire and supplied blood samples. Twelve targeted metabolites were quantitatively analyzed by gas chromatography/tandem mass spectrometry. Odds ratios (OR) and their corresponding 95% confidence intervals (CI) were calculated using conditional logistic regression models. Statistically significant P-value was defined as P < .05. Increasing 1,5-anhydro-d-glucitol (1,5-AG) levels were associated with a decreasing trend in pancreatic cancer risk (OR of quartile 4 [Q4], 0.50; 95% CI, 0.27-0.93; P = .02). Increasing methionine levels were also associated with an increasing trend of pancreatic cancer risk (OR of Q4, 1.79; 95% CI, 0.94-3.40: P = .03). Additional adjustment for potential confounders attenuated the observed associations of 1,5-AG and methionine (P for trend = .06 and .07, respectively). Comparing subjects diagnosed in the first 0-6 years, higher levels of 1,5-AG, asparagine, tyrosine and uric acid showed a decreasing trend for pancreatic cancer risk (P for trend = .04, .04, .04 and .02, respectively), even after adjustment for potential confounders. We found that the 12 target metabolites were not associated with pancreatic cancer risk. However, metabolic changes in the subjects diagnosed in the first 0-6 years showed a similar tendency to our previous reports. These results might suggest that these metabolites are useful for early detection but not for prediction of pancreatic cancer.

We identified two new variants in the third exon of the α-globin gene in families from southern Italy: the Hb Rogliano, α1 cod108 ACC>AAC or α1[α108(G15)Thr→Asn] and the Hb Policoro, α2 cod124 TCC>CCC or α2[α124(H7)Ser→Pro]. The carriers showed mild α-thalassemia phenotype and abnormal hemoglobin stability features. These mutations occurred in the G and H helices of the α-globin both involved in the specific recognition of AHSP and β1 chain. Molecular characterization of mRNA, globin chain analyses and molecular modelling studies were carried out to highlight the mechanisms causing the α-thalassemia phenotype. The results demonstrated that the α-thalassemia defect associated with the two Hb variants originated by different defects. Hb Rogliano showed an intrinsic instability of the tetramer due to anomalous intra- and inter-chain interactions suggesting that the variant chain is normally synthesized and complexed with AHSP but rapidly degraded because it is unable to form the α1β1 dimers. On the contrary in the case of Hb Policoro two different molecular mechanisms were shown: the reduction of the variant mRNA level by an unclear mechanism and the protein instability due to impairment of AHSP interaction. These data highlighted that multiple approaches, including mRNA quantification, are needed to properly identify the mechanisms leading to the α-thalassemia defect. Elucidation of the specific mechanism leads to the definition of a given phenotype providing important guidance for the diagnosis of unstable variants.

This work describes a dysfibrinogenemia linked to a new mutation in the gene coding for fibrinogen γ chain. Dysfibrinogenemia was fortuitously discovered in a 9-year old boy consulting for symptoms suggesting meningitis. DNA was extracted from blood, the fibrinogen genes coding for Aα, Bβ and γ chains were sequenced, and compared with consensus sequences. Apart from the patient, dysfibrinogenemia and the mutation p.H103N in the γ chain of fibrinogen with heterozygous status were found in his mother, without any symptom. This mutation is unknown in fibrinogen variant databases and seems to affect mostly fibrin polymerisation. The reporting of this new p.H103N mutation in the γ chain has a great interest for improving the knowledge of the fibrinogen gene and its expression. Even if no haemorrhage was observed in this case, the expression of this mutation impaired the function of the molecule, particularly polymerisation, and could induce bleeding during an important surgery.

BACKGROUND: Asparagine N-Glycosylation is one of the most important forms of protein post-translational modification in eukaryotes. This metabolic pathway can be subdivided into two parts: an upstream sub-pathway required for achieving proper folding for most of the proteins synthesized in the secretory pathway, and a downstream sub-pathway required to give variability to trans-membrane proteins, and involved in adaptation to the environment and innate immunity. Here we analyze the nucleotide variability of the genes of this pathway in human populations, identifying which genes show greater population differentiation and which genes show signatures of recent positive selection. We also compare how these signals are distributed between the upstream and the downstream parts of the pathway, with the aim of exploring how forces of population differentiation and positive selection vary among genes involved in the same metabolic pathway but subject to different functional constraints.
RESULTS: Our results show that genes in the downstream part of the pathway are more likely to show a signature of population differentiation, while events of positive selection are equally distributed among the two parts of the pathway. Moreover, events of positive selection are frequent on genes that are known to be at bifurcation points, and that are identified as being in key position by a network-level analysis such as MGAT3 and GCS1.
CONCLUSIONS: These findings indicate that the upstream part of the Asparagine N-Glycosylation pathway has lower diversity among populations, while the downstream part is freer to tolerate diversity among populations. Moreover, the distribution of signatures of population differentiation and positive selection can change between parts of a pathway, especially between parts that are exposed to different functional constraints. Our results support the hypothesis that genes involved in constitutive processes can be expected to show lower population differentiation, while genes involved in traits related to the environment should show higher variability. Taken together, this work broadens our knowledge on how events of population differentiation and of positive selection are distributed among different parts of a metabolic pathway.

Acrylamide concentrations in prune products--baby strained prunes (range = 75-265 µg kg(-1)), baby apple/prune juice (33-61 µg kg(-1)), prune juice (186-916 µg kg(-1)) and prunes (58-332 µg kg(-1))--on the Canadian market were determined. The formation of acrylamide in a simulated plum juice was also investigated under \'drying conditions\' in an open vessel at temperatures <100°C for 24 h and under \'wet conditions\' in a closed vessel at a temperature of 120°C for 1 h. Acrylamide was produced in a simulated plum juice under \'drying conditions\' in amounts comparable with those found in prunes and prune juices. Acrylamide was not produced in simulated plum juice under \'wet conditions\' in a closed vessel at temperature of 120°C for 1 h, but under the same condition an authentic prune juice doubled its acrylamide concentration. Formation of acrylamide in prune products was attributed to the presence of asparagine and sugars in the starting materials.

The article describes paraganglioma case in woman with von Hippel-Lindau disease. She was found to be a carrier of a rare germline mutation in the VHL gene (393C>A; N131K). The patient developed large, untypical for von Hippel-Lindau disease, carotid body paraganglioma at the common carotid artery bifurcation. The carotid body paraganglioma coexisted with the haemangioblastoma situated intramedullary in region C5/C6. The haemangioblastoma reached the right-sided dorsal part of the spinal cord in section C5/C6. It produced radicular symptoms within C5/C6, followed by the later paresis of the right limbs. The haemangioblastoma was resected completely. Twelve months after the operation, the spinal symptoms receded and the carotid body paraganglioma still was asymptomatic. The current case of carotid body paraganglioma in patient with the 393C>A (N131K) missense mutation in the VHL gene, supports association of this specific mutation and VHL disease type 2, and suggests its correlation with susceptibility to paragangliomas.