Structure-function relationships in histidine-rich antimicrobial peptides from Atlantic cod.

Gad-1 and Gad-2 are antimicrobial peptide (AMP) sequences encoded by paralogous genes. They are rich in histidine, which suggests that their activity might be pH-dependent. We examined their structure-function relationships with a view to learning how to improve AMP therapeutic ratios. Activity assays with Gram-negative bacteria and cancer cell lines demonstrate that Gad-2 is substantially more active at slightly acidic pH than it is at neutral pH. By contrast, the activity of Gad-1 at lower pH is similar to its activity at pH7. Circular dichroism spectra indicate that the greater functional plasticity of Gad-2 correlates with a greater structural plasticity; Gad-2's percent helicity varies dramatically with altered pH and lipid environment. Interestingly, Gad-2's highest levels of helicity do not correspond to the conditions where it is most active. High resolution solution NMR structures were determined in SDS micelles at pH5, conditions that induce an intermediate level of helicity in the peptides. Gad-1 is more helical than Gad-2, with both peptides exhibiting the greatest helical tendencies in their central region and lowest helicity in their N-termini. The high resolution structures suggest that maximum activity relies on the appropriate balance between an N-terminal region with mixed hydrophobic/hydrophilic structure features and an amphipathic central and C-terminal region. Taken together with previous studies, our results suggest that to improve the therapeutic ratio of AMPs, consideration should be given to including sequential histidine-pairs, keeping the overall charge of the peptide modest, and retaining a degree of structural plasticity and imperfect amphipathicity.

Impact of asymptomatic nodavirus carrier state and intraperitoneal viral mimic injection on brain transcript expression in Atlantic cod (Gadus morhua).

Nodaviruses and other RNA viruses have a profoundly negative impact on the global aquaculture industry. Nodaviruses target nervous tissue causing viral nervous necrosis, a disease characterized by neurological damage, swimming abnormalities, and morbidity. This study used functional genomic techniques to study the Atlantic cod (Gadus morhua) brain transcript expression responses to asymptomatic high nodavirus carrier state and intraperitoneal injection of polyriboinosinic polyribocytidylic acid (pIC). Reciprocal suppression subtractive hybridization (SSH) cDNA libraries enriched for virus-responsive brain transcripts were constructed and characterized. We generated 1,938 expressed sequence tags (ESTs) from a forward brain SSH library (enriched for transcripts upregulated by nodavirus and/or pIC) and 1,980 ESTs from a reverse brain SSH library (enriched for transcripts downregulated by nodavirus and/or pIC). To examine the effect of nodavirus carrier state on individual brain gene expression in asymptomatic cod, 27 transcripts of interest were selected for quantitative reverse transcription-polymerase chain reaction (QPCR) studies. Transcripts found to be >10-fold upregulated in individuals with a high nodavirus carrier state relative to those in a no/low nodavirus carrier state were identified as ISG15, IL8, DHX58 (alias LGP2), ZNFX1, RSAD2 (alias viperin), and SACS (sacsin, alias spastic ataxia of Charlevoix-Saguenay). These and other SSH-identified transcripts were also found by QPCR to be significantly (P < 0.05) upregulated by pIC compared with saline-injected controls within 72 h of injection. Several transcripts identified in the reverse SSH library, including two putative ubiquitination pathway members (HERC4 and SUMO2), were found to be significantly (P < 0.05) downregulated in individuals with a high nodavirus carrier state. Our data shows that Atlantic cod brains have a strong interferon pathway response to asymptomatic high nodavirus carrier state and that many interferon pathway and other immune relevant transcripts are significantly induced in brain by both nodavirus and pIC.

Association of Gabapentin or Pregabalin Use and Incidence of Acute Angle-closure Glaucoma.

PRECIS: Gabapentin and its derivatives have numerous indications and are commonly prescribed medications. In this article, we provide evidence of a link between gabapentinoid use and incidence of acute angle-closure glaucoma. PURPOSE: Gabapentinoids, such as gabapentin and pregabalin, are commonly prescribed classes of drugs in North America. We sought to determine the association of gabapentin or pregabalin use and the incidence of acute angle-closure glaucoma. MATERIALS AND METHODS: This was a nested case-control study. All adult patients who developed acute angle-closure glaucoma between January 1, 2006 and December 31, 2016, and enrolled in the PharMetrics Plus database were eligible for inclusion. A conditional logistic regression model was constructed to assess the association between gabapentin or pregabalin use and the incidence of acute angle-closure glaucoma. RESULTS: Incidence of acute angle-closure glaucoma was found to be statistically significantly associated with the use of gabapentin in the year before diagnosis [relative risk (RR), 1.42; 95% confidence interval (CI), 1.00-2.00]. This association was not observed to be statistically significant with the current use of gabapentin (RR, 1.28; 95% CI, 0.77-2.12). Incidence of acute angle-closure glaucoma (AAG) was not found to be statistically significantly associated with either use of pregabalin in the year before diagnosis or current use (RR, 1.00; 95% CI, 0.51-1.93 and RR, 1.50; 95% CI, 0.66-3.38, respectively). CONCLUSIONS: To the best of our knowledge this is the first study to investigate the association between gabapentin or pregabalin use and the incidence of AAG. Gabapentin use in the year before diagnosis was found to be associated with the incidence of AAG.

Characterization and expression studies of Gaduscidin-1 and Gaduscidin-2; paralogous antimicrobial peptide-like transcripts from Atlantic cod (Gadus morhua).

Piscidins are a family of antimicrobial peptides (AMPs) from fish that constitute an important component of their innate immune system. Based on previously generated Atlantic cod (Gadus morhua) expressed sequence tags (ESTs), we identified sequences representing two paralogous AMP-like transcripts. These Atlantic cod paralogues were termed gaduscidins (GAD-1 and GAD-2), derived from the genus name Gadus. We obtained full-length cDNA sequences for these putative AMP-encoding transcripts using bi-directional rapid amplification of cDNA ends (RACE). GAD-1 and GAD-2 putative peptides exhibit sequence similarity with members of the piscidin family from teleost fish. Quantitative reverse transcription - polymerase chain reaction (QPCR) was utilized in transcript expression studies of GAD-1 and GAD-2. We examined the constitutive expression of these transcripts in six tissues (head kidney, blood, brain, gill, pyloric caecum, and spleen) of non-stressed juvenile cod; GAD-1 and GAD-2 transcripts were detected in all six tissues, with the highest expression of both transcripts being in spleen, head kidney, and gill. Transcript expression of GAD-1 and GAD-2 was also examined in immune tissues (spleen and head kidney) following intraperitoneal (IP) injection of formalin-killed, atypical Aeromonas salmonicida (ASAL) or phosphate-buffered saline (PBS control). Both transcripts were weakly (less than 4-fold) up-regulated by ASAL in spleen but non-responsive to ASAL in head kidney. Since GAD-1 and GAD-2 transcripts are highly expressed constitutively in immune-relevant tissues (e.g. spleen and head kidney), and are weakly induced in spleen following IP injection with bacterial antigens, they may represent important components of innate immunity in Atlantic cod.