Peptide Transporter Research Articles

Absence of the Peptide Transporter 1 Induces a Prediabetic and Depressive-Like Phenotype in Mice.

Protein-enriched diets improve glycemic control in diabetes or emotional behavior in depressive patients. In mice, these benefits depend on intestinal gluconeogenesis activation by di-/tripeptides. Intestinal di-/tripeptides absorption is carried out by the peptide transporter 1, PEPT1. The lack of PEPT1 might thus alter glucose and emotional balance. To determine the effects of PEPT1 deficiency under standard dietary conditions or during a dietary challenge known to promote both metabolic and cognitive dysfunction, insulin sensitivity, anxiety, and depressive-like traits, hippocampal serotonin (5-HT) and insulin signaling pathway were measured in wild-type (WT) and Pept1-/- mice fed either a chow or a high-fat high-sucrose (HF-HS) diet. Pept1-/- mice exhibited slight defects in insulin sensitivity and emotional behavior, which were aggravated by an HF-HS diet. Pept1-/- mice fed a chow diet had lower hippocampal 5-HT levels and exhibited cerebral insulin resistance under HF-HS diet. These defects were independent of intestinal gluconeogenesis but might be linked to increased plasma amino acids levels. Pept1-/- mice develop prediabetic and depressive-like traits and could thus be used to develop strategies to prevent or cure both diseases.

Interdomain communication in a homodimeric ABC transporter

ABC transporters are found in all organisms and almost every cellular compartment. They mediate the transport of various solutes across membranes, energized by ATP binding and hydrolysis. Dysfunctions can result in severe diseases, such as cystic fibrosis or antibiotic resistance. In type IV ABC transporters, each of the two nucleotide-binding domains is connected to a transmembrane domain by two coupling helices, which are part of cytosolic loops. Although there are many structural snapshots of different conformations, the interdomain communication is still enigmatic. Therefore, we analyzed the function of three conserved, charged residues in the intra-cytosolic loop 1 of the human homodimeric, lysosomal peptide transporter TAPL. Substitution of D278 in coupling helix 1 by alanine interrupted peptide transport by impeding ATP hydrolysis. Alanine substitution of R288 and D292, both localized next to the coupling helix 1 extending to transmembrane helix 3, reduced peptide transport but increased basal ATPase activity. Surprisingly, the ATPase activity of the R288A variant dropped in a peptide-dependent manner while ATPase activity of wildtype and D292A was unaffected. Interestingly, R288A and D292A mutants did not differentiate between ATP and GTP in respect of hydrolysis. However, in contrast to wildtype TAPL, only ATP energized peptide transport. In sum, D278 seems to be involved in bidirectional interdomain communication mediated by network of polar interactions while the two residues in the cytosolic extension of TMH3 are involved in regulation of ATP hydrolysis, most likely by stabilization of the outward facing conformation.

The Relationship of Mitochondrial-derived Peptide (MOTS-c) and Brain Mitochondrial Carrier Protein 1 (BMCP1) Response in Sheep with Some Physiological Parameters

Background: Mitochondria are organelles called power plants of the cell, which are found in eukaryotic cells and are responsible for the production of energy needed by the cells through aerobic respiration. Mitochondrial dysfunction causes endocrinological, cardiovascular, metabolic and neurodegenerative diseases. The aim of this study was to examine the effects of body condition score (BCS), lactation, pregnancy, age and gender on Mitochondrial-Derived Peptide (MOTS-c) and Brain Mitochondrial Transporter Protein 1 (BMCP1) levels in Akkaraman sheep. Methods: Sheep are classified into 3 categories according to BCS (BCS less than 2, BCS=3-3.5, BCS≥4). In creating groups in terms of BCS, it was determined by the palpation method applied to the waist area. The sheep included in the study were divided into 4 groups: early lactation, early pregnancy, late pregnancy (dry period) and age (female and male sheep).1st group, ewes in the first period of pregnancy (on the 100th day of the first pregnancy) (n = 30), 2nd group, ewes in the early lactation period (n = 30), 3rd group, ewes in the dry period (the last two months of pregnancy) (n = 30) and in the 4th group, less than 2 (young) (n=30), 2-8 (adult) (n=30), 8 greater than (old) (n=30), male and female sheep (n=30) of different ages. =180) total of 270 animals. Blood samples were taken from the jugular vein of the sheep before the morning feeding. MOTSc and BMCP1 levels were determined in blood serum samples by ELISA method. Result: In the study, serum MOTS-c and BMCP1 levels in sheep during lactation and pregnancy were determined to be lowest in the VKS less than 2 group and highest in the VKS≥4 groups. Likewise, in age and gender groups, the highest level was detected in young lambs and yearlings in the VKS less than 2 group, while the highest level was detected in old sheep and rams in the VKS≥4 group. As a result, it was determined that different age, gender, pregnancy and lactation periods affected serum MOTS-c and BMCP1 levels depending on the change in BCS (P less than 0.05). It was concluded that MOTS-c and BMCP1 may be useful parameters in the evaluation of mitochondrial function, energy metabolism and metabolic profile.

Structural basis for antibiotic transport and inhibition in PepT2, the mammalian proton-coupled peptide transporter.

The uptake and elimination of beta-lactam antibiotics in the human body are facilitated by the proton-coupled peptide transporters PepT1 (SLC15A1) and PepT2 (SLC15A2). The mechanism by which SLC15 family transporters recognize and discriminate between different drug classes and dietary peptides remains unclear, hampering efforts to improve antibiotic pharmacokinetics through targeted drug design and delivery. Here, we present cryo-EM structures of the mammalian proton-coupled peptide transporter, PepT2, in complex with the widely used beta-lactam antibiotics cefadroxil, amoxicillin and cloxacillin. Our structures, combined with pharmacophore mapping, molecular dynamics simulations and biochemical assays, establish the mechanism of antibiotic recognition and the important role of protonation in drug binding and transport.

Effects of probiotic supplementary bioflocs (Rhodospirillum rubrum, Bacillus subtilis, Providencia rettgeri) on growth, immunity, and water quality in cultures of Pacific white shrimp (Litopenaeus vannamei)

Biofloc technology is a promising strategy for the super-intensive cultivation of Pacific white shrimp (Litopenaeus vannamei). However, conventional bioflocs originating from pre-existing systems have shown to be inferior to bioflocs with constant probiotic supplementation regarding the stability in yield. Hence we compared bioflocs supplemented with Rhodospirillum rubrum (RR), Bacillus subtilis (BS), and Providencia rettgeri (PR) to the clear water system (CT), in terms of growth performances, immune status, nitrogen substances, and Vibrio populations in cultures of L. vannamei together with microbial profiles in both the midgut and bioflocs. Our findings exhibited increased average daily gain (ADG) and protein efficiency ratio (PER) among shrimp reared in BS and PR while BS also reduced feed conversion ratio (FCR). Gene expression analyses of growth-related markers revealed an upregulation of amylase and peptide transporter 1 in shrimp of PR treatment, whilst the changes did not outperform the BS supplement in growth promotion. Furthermore, PR treatment was found to enhance the expression of immune-related genes toll-like receptor, prophenoloxidase (proPO), and heat shock protein 70 (hsp70). All biofloc treatments effectively reduced NO2-N level and mitigated Vibrio parahaemolyticus proliferation, particularly in the BS and PR groups. The redundant analysis illustrated shrimp growth closely associated with improved water quality instead of the expression of genes participating in digestion, absorption, or immunity. Moreover, microflora sequencing exhibited the supplemental probiotics altered microbial communities in bioflocs and gut, whereas they did not dominate in either environment. In spite of the discrepancies in microbial composition between bioflocs and midgut of shrimp, BS and PR treatments enriched Rhodobacteraceae populations in both environments. In summary, B. subtilis and P. rettgeri supplementary bioflocs promote L. vannamei growth through nutritional supplementation and water environment optimization. Supplementation of P. rettgeri will simultaneously enhance immune responses and stress resistance of L. vannamei via activating proPO cascade and upregulating hsp70. Given the apparent benefits of heterotrophic probiotic supplementary bioflocs, it is imperative to investigate more microbial species or their combinations that can stimulate bioflocs to produce more nutrients.

Investigating the Transepithelial Transport and Enzymatic Stability of Lactononadecapeptide (NIPPLTQTPVVVPPFLQPE), a 19-Amino Acid Casein-Derived Peptide in Caco-2 Cells.

Lactononadecapeptide (LNDP; NIPPLTQTPVVVPPFLQPE), a casein-derived peptide comprising 19 residues, is known for its capacity to enhance cognitive function. This study aimed to explore the transepithelial transport and stability of LNDP. Results showed that LNDP retained over 90% stability after 2 h of treatment with gastrointestinal enzymes. The stability of LNDP on Caco-2 cell monolayers ranged from 93.4% ± 0.9% to 101.1% ± 1.2% over a period of 15-60 min, with no significant differences at each time point. The permeability of LNDP across an artificial lipid membrane was very low with the effective permeability of 3.6 × 10-11 cm/s. The Caco-2 assay demonstrated that LNDP could traverse the intestinal epithelium, with an apparent permeability of 1.22 × 10-6 cm/s. Its transport was significantly inhibited to 67.9% ± 5.0% of the control by Gly-Pro, a competitor of peptide transporter 1 (PEPT1). Furthermore, PEPT1 knockdown using siRNA significantly inhibited LNDP transport by 77.6% ± 1.9% in Caco-2 cell monolayers. The LNDP uptake in PEPT1-expressing HEK293 cells was significantly higher (54.5% ± 14.6%) than that in mock cells. These findings suggest that PEPT1 plays a crucial role in LNDP transport, and LNDP exhibits good resistance to gastrointestinal enzymes.

Stability and transepithelial transport of oligopeptide (KRQKYD) with hepatocyte-protective activity from Jinhua ham in human intestinal Caco-2 monolayer cells

The study evaluated the stability of an oligopeptide (Lys-Arg-Gln-Lys-Tyr-Asp, KRQKYD) and its transport mechanism by simulating gastrointestinal digestion and a model of human intestinal Caco-2 monolayer cells in vitro. In this study, the effects of environmental factors (temperature, pH and NaCl concentration) and simulated gastrointestinal digestion on the stability of KRQKYD were evaluated by indicators of the levels of alanine transaminase (ALT), aspartate transaminase (AST) and malondialdehyde (MDA) in an alcohol-induced hepatocyte injury model. The results showed that KRQKYD still maintained satisfactory hepatocyte-protective activity after treatment with different temperatures (20−80 °C), pH (3.0−9.0), NaCl concentration (1 %−7 %) and simulated gastrointestinal digestion, which indicated that KRQKYD showed good stability to environmental factors and simulated gastrointestinal digestion. Furthermore, the intact KRQKYD could be absorbed in a model of Caco-2 monolayer cells with a Papp value of (9.70 ± 0.53) × 10−7 cm/s. Pretreatment with an energy inhibitor (sodium azide), a competitive peptide transporter inhibitor (Gly-Pro) and a transcytosis inhibitor wortmannin did not decrease the level of transepithelial KRQKYD transport, indicating that the transport mechanism of KRQKYD was not associated with energy dependent, vector mediated and endocytosis. The tight junction disruptor cytochalasin D significantly increased the level of transepithelial KRQKYD transport (P < 0.05), suggesting that intact KRQKYD was absorbed by paracellular transport.

Chlamydomonas reinhardtii chloroplast factory construction for formate bioconversion

The photosynthetic autotrophic production of microalgae is limited by the effective supply of carbon and light energy, and the production efficiency is lower than the theoretical value. Represented by methanol, C1 compounds have been industrially produced by artificial photosynthesis with a solar energy efficiency over 10%, but the complexity of artificial products is weak. Here, based on a construction of chloroplast factory, green microalgae Chlamydomonas reinhardtii CC137c was modified for the bioconversion of formate for biomass production. By screening the optimal combination of chloroplast transport peptides, the cabII-1 cTP1 fusion formate dehydrogenase showed significant enhancement on the conversion of formate with a better performance in the maintenance of light reaction activity. This work provided a new way to obtain bioproducts from solar energy and CO2 with potentially higher-than-nature efficiency by the artificial-natural hybrid photosynthesis.

Peptide Transporter 1-Mediated Dipeptide Transport Promotes Hepatocellular Carcinoma Metastasis by Activating MAP4K4/G3BP2 Signaling Axis.

Cancer metastasis is the leading cause of mortality in patients with hepatocellular carcinoma (HCC). To meet the rapid malignant growth and transformation, tumor cells dramatically increase the consumption of nutrients, such as amino acids. Peptide transporter 1 (PEPT1), a key transporter for small peptides, has been found to be an effective and energy-saving intracellular source of amino acids that are required for the growth of tumor cells. Here, the role of PEPT1 in HCC metastasis and its underlying mechanisms is explored. PEPT1 is upregulated in HCC cells and tissues, and high PEPT1 expression is associated with poor prognosis in patients with HCC. PEPT1 overexpression dramatically promoted HCC cell migration, invasion, and lung metastasis, whereas its knockdown abolished these effects both in vitro and in vivo. Mechanistic analysis revealed that high PEPT1 expression increased cellular dipeptides in HCC cells that are responsible for activating the MAP4K4/G3BP2 signaling pathway, ultimately facilitating the phosphorylation of G3BP2 at Thr227 and enhancing HCC metastasis. Taken together, these findings suggest that PEPT1 acts as an oncogene in promoting HCC metastasis through dipeptide-induced MAP4K4/G3BP2 signaling and that the PEPT1/MAP4K4/G3BP2 axis can serve as a promising therapeutic target for metastatic HCC.

Understanding the Transepithelial Transport and Transbilayer Diffusion of the Antihypertensive Peptide Asn-Cys-Trp: Insights from Caco-2 Cell Monolayers and the DPPC Model Membrane.

Understanding the transport mechanism of the peptide Asn-Cys-Trp (NCW) is crucial to improving its intestinal absorption and bioavailability. This study investigated the absorption of NCW through Caco-2 cell monolayers and its interaction with the DPPC bilayers. Results revealed that after a 3 h incubation, the Papp (AP-BL) and Papp (BL-AP) values of NCW at a concentration of 5 mmol/L were (22.24 ± 4.52) × 10-7 and (6.63 ± 2.31) × 10-7 cm/s, respectively, with the transport rates of 1.59 ± 0.32 and 0.62 ± 0.20%, indicating its moderate absorption. NCW was found to be transported via PepT1 and paracellular transport pathways, as evidenced by the significant impact of Gly-Pro and cytochalasin D on the Papp values. Moreover, NCW upregulated ZO-1 mRNA expression. Further investigation of the ZO-1-mediated interaction between NCW and tight junction proteins will contribute to a better understanding of the paracellular transport mechanism of NCW. The interaction between NCW and the DPPC bilayers was predominantly driven by entropy. NCW permeated the bilayers through electrostatic, hydrogen bonding, and hydrophobic interactions, resulting in increased fluidity, flexibility, and disorder as well as phase transition and phase separation of the bilayers.

Human ABC and SLC Transporters: The Culprit Responsible for Unspecific PSMA-617 Uptake?

[177Lu]Lu-PSMA-617 has recently been successfully approved by the FDA, the MHRA, Health Canada and the EMA as Pluvicto®. However, salivary gland (SG) and kidney toxicities account for its main dose-limiting side-effects, while its corresponding uptake and retention mechanisms still remain elusive. Recently, the presence of different ATP-binding cassette (ABC) transporters, such as human breast cancer resistance proteins (BCRP), multidrug resistance proteins (MDR1), multidrug-resistance-related proteins (MRP1, MRP4) and solute cassette (SLC) transporters, such as multidrug and toxin extrusion proteins (MATE1, MATE2-K), organic anion transporters (OAT1, OAT2v1, OAT3, OAT4) and peptide transporters (PEPT2), has been verified at different abundances in human SGs and kidneys. Therefore, our aim was to assess whether [177Lu]Lu-PSMA-617 and [225Ac]Ac-PSMA-617 are substrates of these ABC and SLC transporters. For in vitro studies, the novel isotopologue ([α,β-3H]Nal)Lu-PSMA-617 was used in cell lines or vesicles expressing the aforementioned human ABC and SLC transporters for inhibition and uptake studies, respectively. The corresponding probe substrates and reference inhibitors were used as controls. Our results indicate that [177Lu]Lu-PSMA-617 and [225Ac]Ac-PSMA-617 are neither inhibitors nor substrates of the examined transporters. Therefore, our results show that human ABC and SLC transporters play no central role in the uptake and retention of [177Lu]Lu-PSMA-617 and [225Ac]Ac-PSMA-617 in the SGs and kidneys nor in the observed toxicities.

Tuning the double lipidation of salmon calcitonin to introduce a pore-like membrane translocation mechanism

A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it’s membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure–function relations could be pivotal for optimizing future peptide design strategies.

Dietary crude protein and protein solubility manipulation enhances intestinal nitrogen absorption and mitigates reactive nitrogen emissions through gut microbiota and metabolome reprogramming in sheep

Dietary nutrient manipulation (e.g. protein fractions) could lower the environmental footprints of ruminants, especially reactive nitrogen (N). This study investigated the impacts of dietary soluble protein (SP) levels with decreased crude protein (CP) on intestinal N absorption, hindgut N metabolism, fecal microbiota and metabolites, and their linkage with N metabolism phenotype. Thirty-two male Hu sheep, with an age of six months and an initial BW of 40.37 ± 1.18 kg, were randomly assigned to four dietary groups. The control diet (CON), aligning with NRC standards, maintained a CP content of 16.7% on a dry matter basis. Conversely, the experimental diets (LPA, LPB, and LPC) featured a 10% reduction in CP compared with CON, accompanied by SP adjustments to 21.2%, 25.9%, and 29.4% of CP, respectively. Our results showed that low-protein diets led to significant reductions in the concentrations of plasma creatinine, ammonia, urea N, and fecal total short-chain fatty acids (SCFA) (P < 0.05). Notably, LPB and LPC exhibited increased total SCFA and propionate concentrations compared with LPA (P < 0.05). The enrichment of the Prevotella genus in fecal microbiota associated with energy metabolism and amino acid (AA) biosynthesis pathways was evident with SP levels in low-protein diets of approximately 25% to 30%. Moreover, LPB and LPC diets demonstrated a decrease in fecal NH4+–N and NO2−–N contents as well as urease activity, compared with CON (P < 0.05). Concomitantly, reductions in fecal glutamic acid dehydrogenase gene (gdh), nitrite reductase gene (nirS), and nitric oxide reductase gene (norB) abundances were observed (P < 0.05), pointing towards a potential reduction in reactive N production at the source. Of significance, the up-regulation of mRNA abundance of AA and peptide transporters in the small intestine (duodenum, jejunum, and ileum) and the elevated concentration of plasma AA (e.g. arginine, methionine, aspartate, glutamate, etc.) underscored the enhancement of N absorption and N efficiency. In summary, a 10% reduction in CP, coupled with an SP level of approximately 25% to 30%, demonstrated the potential to curtail reactive N emissions through fecal Prevotella enrichment and improve intestinal energy and N utilization efficiency.

Quality by design (QbD) approach-based development of optimized nanocarrier to achieve quality target product profile (QTPP)-targeted lymphatic delivery

Background. Insulin, commonly used for diabetes treatment, needs better ways to improve its effectiveness and safety due to its challenges with poor permeability and stability. Various system has been developed for oral peptide delivery. The non-targeted system can prevent gastric and enzymatic degradation of peptides but cannot increase the bulk transport of peptides across the membrane. However, the non-selectivity is the limitation of the existing system. Numerous carbohydrate-binding receptors overexpressed on intestinal macrophage cells (M-cells) of gut-associated lymphoid tissue. It is the most desirable site for receptor-mediated endocytosis and lymphatic drug delivery of peptides. Objective. The prime objective of the study was to fabricate mannose ligand conjugated nanoparticles (MNPs) employing a quality-by-design approach to address permeability challenges after oral administration. Herein, the study’s secondary objective of this study is to identify the influencing factor for producing quality products. Considering this objective, the Lymphatic uptake of NPs was selected as a quality target product profile (QTPP), and a systematic study was conducted to identify the critical formulation attributes (CFAs) and critical process parameters (CPP) influencing critical quality attributes (CQAs). Mannosylated Chitosan concentrations (MCs) and TPP concentrations were identified as CFAs, and stirring speed was identified as CPP. Methods. MNPs were prepared by the inotropic gelation method and filled into the enteric-coated capsule to protect from acidic environments. The effect of CFAs and CPP on responses like particle size (X) and entrapment (Y) was observed by Box-Behnken design (BBD). ANOVA statistically evaluated the result to confirm a significant level (p < 0.05). The optimal conditions of NPs were obtained by constructing an overlay plot and determining the desirability value. HPLC and zeta-seizer analysis characterized the lyophilized NPs. Cell-line studies were performed to confirm the safety and M-cell targeting of NPs to enhance Insulin oral bioavailability. Results. The morphology of NPs was revealed by SEM. The developed NPs showed a nearly oval shape with the average size, surface potential, and % drug entrapment were 245.52 ± 3.37 nm, 22.12 ± 2.13 mV, and 76.15 ± 1.3%, respectively. MTT assay result exhibited that MNPs safe and Confocal imaging inference that NPs selectively uptake by the M-cell. Conclusion. BBD experimental design enables the effective formulation of optimized NPs. The statistical analysis estimated a clear assessment of the significance of the process and formulation variable. Cell line study confirms that NPs are safe and effectively uptake by the cell.

An oligo peptide transporter family member, OsOPT7, mediates xylem unloading of Fe for its preferential distribution in rice.

Iron (Fe) needs to be delivered to different organs and tissues of above-ground parts for playing its multiple physiological functions once it is taken up by the roots. However, the mechanisms underlying Fe distribution are poorly understood. We functionally characterized OsOPT7, a member of oligo peptide transporter family in terms of expression patterns, localization, transport activity and phenotypic analysis of knockdown lines. OsOPT7 was highly expressed in the nodes, especially in the uppermost node I, and its expression was upregulated by Fe-deficiency. OsOPT7 transports ferrous iron into the cells coupled with proton. Immunostaining revealed that OsOPT7 is mainly localized in the xylem parenchyma cells of the enlarged vascular bundles in the nodes and vascular tissues in the leaves. Knockdown of OsOPT7 did not affect the Fe uptake, but altered Fe distribution; less Fe was distributed to the new leaf, upper nodes and developing panicle, but more Fe was distributed to the old leaves. Furthermore, knockdown of OsOPT7 also resulted in less Fe distribution to the leaf sheath, but more Fe to the leaf blade. Taken together, OsOPT7 is involved in the xylem unloading of Fe for both long-distance distribution to the developing organs and local distribution within the leaf in rice.

Molecular characterization, localization, and physiological roles of ITP and ITP-L in the mosquito, Aedes aegypti.

The insect ion transport peptide (ITP) and its alternatively spliced variant, ITP-like peptide (ITP-L), belong to the crustacean hyperglycemic hormone family of peptides and are widely conserved among insect species. While limited, studies have characterized the ITP/ITP-L signaling system within insects, and putative functions including regulation of ion and fluid transport, ovarian maturation, and thirst/excretion have been proposed. Herein, we aimed to molecularly investigate Itp and Itp-l expression profiles in the mosquito, Aedes aegypti, examine peptide immunolocalization and distribution within the adult central nervous system, and elucidate physiological roles for these neuropeptides. Transcript expression profiles of both AedaeItp and AedaeItp-l revealed distinct enrichment patterns in adults, with AedaeItp expressed in the brain and AedaeItp-l expression predominantly within the abdominal ganglia. Immunohistochemical analysis within the central nervous system revealed expression of AedaeITP peptide in a number of cells in the brain and in the terminal ganglion. Comparatively, AedaeITP-L peptide was localized solely within the pre-terminal abdominal ganglia of the central nervous system. Interestingly, prolonged desiccation stress caused upregulation of AedaeItp and AedaeItp-l levels in adult mosquitoes, suggesting possible functional roles in water conservation and feeding-related activities. RNAi-mediated knockdown of AedaeItp caused an increase in urine excretion, while knockdown of both AedaeItp and AedaeItp-l reduced blood feeding and egg-laying in females as well as hindered egg viability, suggesting roles in reproductive physiology and behavior. Altogether, this study identifies AedaeITP and AedaeITP-L as key pleiotropic hormones, regulating various critical physiological processes in the disease vector, A. aegypti.

A Walk on the Wild Side: Genome Editing of Tuber-Bearing Solanum bulbocastanum.

Solanum bulbocastanum is a wild diploid tuber-bearing plant. We here demonstrate transgene-free genome editing of S. bulbocastanum protoplasts and regeneration of gene-edited plants. We use ribonucleoproteins, consisting of Cas9 and sgRNA, assembled in vitro, to target a gene belonging to the nitrate and peptide transporter family. Four different sgRNAs were designed and we observed efficiency in gene-editing in the protoplast pool between 8.5% and 12.4%. Twenty-one plants were re-generated from microcalli developed from individual protoplasts. In three of the plants we found that the target gene had been edited. Two of the edited plants had deletion mutations introduced into both alleles, whereas one only had a mutation in one of the alleles. Our work demonstrates that protocols for the transformation of Solanum tuberosum can be optimized to be applied to a wild Solanum species.

The Role of MXene Surface Terminations on Peptide Transportation in Nanopore Sensing.

Nanopores with two-dimensional materials have various advantages in sensing, but the fast translocation of molecules hinders their scale-up applications. In this work, we investigate the influence of -F, -O, and -OH surface terminations on the translocation of peptides through MXene nanopores. We find that the longest dwell time always occurs when peptides pass through the Ti3C2O2 nanopores. This elongated dwell time is induced by the strongest interaction between peptides and the Ti3C2O2 membrane, in which the van der Waals interactions dominate. Compared to the other two MXene nanopores, the braking effect is indicated during the whole translocation process, which evidence the advantage of Ti3C2O2 in nanopore sensing. Our work demonstrates that membrane surface chemistry has a great influence on the translocation of peptides, which can be introduced in the design of nanopores for a better performance.

Cellular Uptake of Cell-Penetrating Peptides Activated by Amphiphilic p-Sulfonatocalix[4]arenes.

We report the synthesis of a series of amphiphilic p-sulfonatocalix[4]arenes with varying alkyl chain lengths (CX4-Cn) and their application as efficient counterion activators for membrane transport of cell-penetrating peptides (CPPs). The enhanced membrane activity is confirmed with the carboxyfluorescein (CF) assay in vesicles and by the direct cytosolic delivery of CPPs into CHO-K1, HCT 116, and KTC-1 cells enabling excellent cellular uptake of the CPPs into two cancer cell lines. Intracellular delivery was confirmed by fluorescence microscopy after CPP entry into live cells mediated by CX4-Cn, which was also quantified after cell lysis by fluorescence spectroscopy. The results present the first systematic exploration of structure-activity relationships for calixarene-based counterion activators and show that CX4-Cn are exceptionally effective in cellular delivery of CPPs. The dodecyl derivative, CX4-C12, serves as best activator. A first mechanistic insight is provided by efficient CPP uptake at 4 °C and in the presence of the endocytosis inhibitor dynasore, which indicates a direct translocation of the CPP-counterion complexes into the cytosol and highlights the potential benefits of CX4-Cn for efficient and direct translocation of CPPs and CPP-conjugated cargo molecules into the cytosol of live cells.

Replacing fishmeal with salmon hydrolysate reduces the expression of intestinal inflammatory markers and modulates the gut microbiota in Atlantic salmon (Salmo salar)

The feed legislation allows the use of fish protein hydrolysates in feed for the same species in which it came from, since enzymatic hydrolysis degrades the proteins and eliminates potential prions, which have caused disease in mammals, but not in fish. In this trial, we investigated the effects of partially replacing dietary fishmeal (FM) with salmon protein hydrolysate (FPH) on the intestinal gene expression and microbiota. Atlantic salmon post smolts were either fed a control diet containing 30% fishmeal (FM), a 20% FM diet with 9% salmon hydrolysate (FPH-09) or a 10% FM diet with 18% salmon hydrolysate (FPH-18), until doubling of weight. Gene expression analysis by RNA sequencing of pyloric caeca (PC), midgut (MG) and hindgut (HG) revealed a downregulation of immunological genes involved in inflammation in the intestine of FPH-18 fed salmon compared to salmon fed the FM control. The gene expression of paralogous peptide transporters (PepT) was analyzed by real time quantitative PCR in PC, anterior midgut (AMG), posterior midgut (PMG) and HG of salmon fed all the three diets. The PepT1b paralog had highest relative expression levels in PC and AMG, suggesting that PepT1b is most important for peptide uptake in the anterior intestine. PepT1a was also mainly expressed in the PC and AMG, but at lower levels than PepT1b and PepT2b in the AMG. The PepT2b paralog had high levels of expression in AMG, PMG and HG indicating that it contributed significantly to peptide uptake in the posterior part of the gastrointestinal tract. The gut microbiota in the mucosa and digesta of the MG and HG, were dominated by the phyla Cyanobacteria and Proteobacteria, but also Firmicutes were present. The only dietary effect on the microbiota was the higher prevalence of the phyla Spirochaetes in the mucosa of FPH-18 fed salmon compared to the FM fed salmon. In conclusion, replacing FM with salmon hydrolysate reduced the expression of inflammatory markers in the Atlantic salmon intestine suggesting improved health benefits. The reduced inflammation may be related to the reduced FM content, potentially bioactive peptides in the hydrolysate and/or the altered gut microbial composition.