Albumins Research Articles

Deciphering the mechanism underlying poor aqueous solubility of extracted quinoa proteins

This study aimed to decipher the mechanisms underlying poor solubility of quinoa proteins by investigating the form of quinoa proteins dispersed in water and how protein-protein interactions influenced the kinetic stability of proteins in the dispersions. Specifically, the relative solubility and the forms of quinoa proteins in 1–5 w/w% protein dispersions were determined by separating proteins via centrifugation and/or ultrafiltration. The kinetic stability of quinoa proteins in the supernatants over a 3-week storage period was characterized by determining the changes of concentration, composition and physicochemical properties of quinoa proteins and predicting protein-protein interactions. The results showed that quinoa proteins existed mainly as differently-sized protein aggregates in the dispersions, leading to low relative solubility. The coagulation of protein aggregates in the supernatants caused severe precipitation during the first week of storage whereas they were disassociated simultaneously. With further storage, the remaining proteins in the supernatants reached kinetic stability, which was contributed by stronger electrostatic repulsion and lower surface hydrophobicity. Moreover, 11S globulin and 2S albumin were precipitated and solubilized together during storage, which was ascribed to intermolecular interactions driven by multiple sites between 11S globulin and/or 2S albumin. This study lays a foundation for extensive utilization of quinoa proteins.

Exploring the biodiversity of plant proteins for sustainable foods: Composition and emulsifying properties of the proteins recovered by aqueous extraction from camelina (Camelina sativa L.) seeds

The food transition towards an increased consumption of plant proteins aimed at limiting environmental impacts requires a diversification of plant protein sources. In this study, we explored the potentialities of the sustainable oilseed crop camelina to provide dietary proteins and to prepare oil-in-water emulsions. An innovative green refinery process, including the removal by ultrasound of the mucilage attached at the surface of the seeds and extraction by grinding in water at pH 8, was used to recover aqueous extracts containing camelina seed proteins. These proteins, mainly composed of the 11S globulin cruciferins and the 2S albumin napins, contained the nine essential amino acids of nutritional interest. Camelina seed proteins exhibited low solubility in the range of pH H 5.5-2, attributed to the cruciferins. Oil-in-water emulsions stabilized by camelina seed proteins revealed the preferential adsorption of napin at the oil/water interface and exhibited physical instability below pH 7. Homogenization of the whole aqueous extracts containing oil bodies (OBs) induced the adsorption of cruciferins and napins together with oleosins at the surface of homogenized OBs, and their sedimentation below pH 6.5. Camelina seed proteins adsorbed at the oil/water interface governed the surface properties of the oil droplets and homogenized OBs and the physical stability of the emulsions as a function of pH. This work brings new insights into the potentialities of camelina seed proteins recovered by aqueous extraction that may serve as natural plant-based functional ingredients in the food industry and that will contribute to the development of innovative and sustainable plant-based food emulsions.

Live-Cell Identification of Inhibitors of the Lipid Transfer Protein CERT Using Nanoluciferase Bioluminescence Resonance Energy Transfer (NanoBRET).

A BRET system is described, in which Nanoluciferase was fused to the lipid transfer protein CERT for efficient energy transfer to a Nile red-labeled ceramide, which is either directly bound to CERT or transported to the adjacent Golgi membrane. Bulk formation of sphingomyelin, a major plasma membrane component in mammals, is dependent on CERT-mediated transfer of its predecessor ceramide. CERT is considered a promising drug target but no direct cell-based methods exist to efficiently identify inhibitors. The utility off the method was demonstrated by a library of 140 derivatives of the CERT inhibitor HPA-12. These were obtained in a combinatorial synthesis using solid-phase transacylation. Screening of the library led to six compounds that were picked and confirmed to be superior to HPA-12 in a subsequent dose-response study and also in an orthogonal lipidomics analysis.

Making lipids very unhappy to discover how they bind to proteins.

Membrane lipid composition is maintained by conserved lipid transfer proteins, but computational approaches to study their lipid-binding mechanisms are limiting. Srinivasan et al. (https://doi.org/10.1083/jcb.202312055) develop a clever molecular dynamics simulations assay to accurately model lipid-binding poses in lipid transfer proteins.

Characterization of two Plasmodium falciparum lipid transfer proteins of the Sec14/CRAL-TRIO family

Invasion of human red blood cells by the malaria parasite Plasmodium falciparum is followed by dramatic modifications of erythrocytes properties, including de novo formation of new membrane systems. Lipid transfer proteins from both the parasite and the host cell are most likely an important part of those membrane remodeling processes. Using bioinformatics and in silico structural analysis, we have identified five P. falciparum potential lipid transfer proteins containing cellular retinaldehyde binding – triple functional domain (CRAL-TRIO). Two of these proteins, C6KTD4, encoded by the PF3D7_0629900 gene and Q8II87, encoded by the PF3D7_1127600 gene, were studied in more detail. In vitro lipid transfer assays using recombinant C6KTD4 and Q8II87 confirmed that these proteins are indeed bona fide lipid transfer proteins. C6KTD4 transfers sterols, phosphatidylinositol 4,5 bisphosphate, and, to some degree, also phosphatidylcholine between two membrane compartments. Q8II87 possesses phosphatidylserine transfer activity in vitro. In the yeast model, the expression of P. falciparumQ8II87 protein partially complements the absence of Sec14p and its closest homologue, Sfh1p. C6KTD4 protein can substitute for the collective essential function of oxysterol-binding related proteins. According to published whole genome studies in P. falciparum, absence of C6KTD4 and Q8II87 proteins has severe consequences for parasite viability. Therefore, CRAL-TRIO lipid transfer proteins of P. falciparum are potential targets of novel antimalarials, in search for which the yeast model expressing these proteins could be a valuable tool.

Bridge-like lipid transfer protein 3A (BLTP3A) is associated with membranes of the late endocytic pathway and is an effector of CASM.

Recent studies have identified a family of rod-shaped proteins which includes VPS13 and ATG2 and are thought to mediate unidirectional lipid transport at intracellular membrane contacts by a bridge-like mechanism. Here, we show that one such protein, BLTP3A/UHRF1BP1, associates with VAMP7-positive vesicles via its C-terminal region and anchors them to lysosomes via the binding of its chorein domain containing N-terminal region to Rab7. Upon damage of lysosomal membranes and resulting mATG8 recruitment to their surface by CASM, BLTP3A first dissociates from lysosomes but then reassociates with them via an interaction of its LIR motif with mATG8. Such interaction is mutually exclusive to the binding of BLTP3A to vesicles and leaves its N-terminal chorein domain, i.e. the proposed entry site of lipids into this family of proteins, available for binding to another membrane, possibly the ER. Our findings reveal that BLTP3A is an effector CASM, potentially as part of a mechanism to help repair or minimize lysosome damage by delivering lipids.

Insights into the functional mechanism of the non-specific lipid transfer protein nsLTP in Kalanchoe fedtschenkoi (Lavender scallops)

Plant non-specific lipid transfer protein (nsLTP) is able to bind and transport lipids and essential oils, as well as engage in various physiological processes, including defense against phytopathogens. Kalanchoe fedtschenkoi (Lavender Scallops) is an attractive and versatile succulent. To investigate the functional mechanism of Kalanchoe fedtschenkoi nsLTP (Ka-nsLTP), we expressed, purified and successfully obtained monomeric Ka-nsLTP. Mutational experiments revealed that the C6A variant retained the same activity as the wild-type (WT) Ka-nsLTP. Ka-nsLTP showed weak antiphytopathogenic bacterial activity, but inhibited fungal growth. Ka-nsLTP possessed a hydrophobic cavity effectively binding lauric acid. Our results offer novel molecular insights into the functional mechanism of nsLTP, which broadens our knowledge of the biological function of nsLTP in crops and provides a useful locus for genetic improvement of plants.

Polar-localized OsLTPG22 regulates rice leaf cuticle deposition and drought response

The cuticle serves as a crucial protective barrier for plant survival, and recent studies have highlighted the essential roles of nonspecific lipid transfer proteins (nsLTPs) in cuticle formation. However, the specific function of nsLTPs in the rice leaf cuticle remains unclear. In this study, we functionally characterized OsLTPG22, a G-type nsLTP with a signal peptide (SP) domain and a glycosylphosphatidylinositol (GPI) anchor region. Mutation in OsLTPG22 led to a reduction in cuticular wax abundance, increased leaf epidermal permeability, and higher drought sensitivity in seedlings. OsLTPG22 was widely expressed in various tissues and exhibited distinct polar localization to the aerial surface of epidermal cells in expanding leaves. OsLTPG22 binds lipids and localizes to the plasma membrane. Protein truncation experiments demonstrated that OsLTPG22’s polar localization was regulated by the SP domain, while both the SP domain and GPI anchor region regulated OsLTPG22’s plasma membrane localization. This work provides genetic and cytological evidence for OsLTPG22’s role in leaf cuticle formation and drought response, enhancing our understanding of nsLTP function and offering insights for breeding drought-resistant crops.

Application of CRISPR/Cas9 system to knock out GluB gene for developing low glutelin rice mutant

The nutritional quality improvement is among the most integral objective for any rice molecular breeding programs. The seed storage proteins (SSPs) have greater role to determine the nutritional quality of any cereal grains. Rice contains relatively balanced amino acid composition and the SSPs are fractioned into albumins (ALB), globulins (GLO), prolamins (PRO) and glutelins (GLU) according to differences in solubility. GLUs are further divided into subfamilies: GluA, GluB, GluC, and GluD depending on resemblance in amino acid. The GLU protein accounts for 60–80% of total protein contents, encoded by 15 genes located on different chromosomes of rice genome. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system was employed to knockout Glu-B (LOC-Os02g15070) gene in non-basmati rice PK386 cultivar. The mutant displayed two base pair and three base pair mutation in the targeted regions. The homozygous mutant plant displayed reduction for both in total protein contents and GLU contents whereas, elevation in GLO, ALB and PRO. Moreover, the mutant plant also displayed reduction in physio-chemical properties e.g., total starch, amylose and gel consistency. The agronomic characteristics of both mutant and wild type displayed non-significant differences along with increase in higher percentage of chalkiness in mutant plants. The results obtained from scanning electron microscopy showed the loosely packed starch granules compared to wild type. The gene expression analysis displayed the lower expression of gene at 5 days after flowering (DAF), 10 DAF, 15 DAF and 20 DAF compared to wild type. GUS sub-cellular localization showed the staining in seed which further validated the results obtained from gene expression. Based on these findings it can be concluded Glu-B gene have significant role in controlling GLU contents and can be utilized in breeding programs to enhance the nutritional quality of rice, and may serve as healthy diet for patient allergic with high GLU contents.

The Roadmap of Plant Antimicrobial Peptides Under Environmental Stress: From Farm to Bedside.

Antimicrobial peptides (AMPs) are the most favorable alternatives in overcoming multidrug resistance, alone or synergistically with conventional antibiotics. Plant-derived AMPs, as cysteine-rich peptides, widely compensate the pharmacokinetic drawbacks of peptide therapeutics. Compared to the putative genes encrypted in the genome, AMPs that are produced under stress are active forms with the ability to combat resistant microbial species. Within this study, plant-derived AMPs, namely, defensins, nodule-specific cysteine-rich peptides, snakins, lipid transfer proteins, hevein-like proteins, α-hairpinins, and aracins, expressed under biotic and abiotic stresses, are classified. We could observe that while α-hairpinins and snakins display a helix-turn-helix structure, conserved motif patterns such as β1αβ2β3 and β1β2β3 exist in plant defensins and hevein-like proteins, respectively. According to the co-expression data, several plant AMPs are expressed together to trigger synergistic effects with membrane disruption mechanisms such as toroidalpore, barrel-stave, and carpet models. The application of AMPs as an eco-friendly strategy in maintaining agricultural productivity through the development of transgenes and bio-pesticides is discussed. These AMPs can be consumed in packaging material, wound-dressing products, coating catheters, implants, and allergology. AMPs with cell-penetrating properties are verified for the clearance of intracellular pathogens. Finally, the dominant pharmacological activities of bioactive peptides derived from the gastrointestinal digestion of plant AMPs, namely, inhibitors of renin and angiotensin-converting enzymes, dipeptidyl peptidase IV and α-glucosidase inhibitors, antioxidants, anti-inflammatory, immunomodulating, and hypolipidemic peptides, are analyzed. Conclusively, as phytopathogens and human pathogens can be affected by plant-derived AMPs, they provide a bright perspective in agriculture, breeding, food, cosmetics, and pharmaceutical industries, translated as farm to bedside.

Complex of Defense Polypeptides of Wheatgrass (Elytrigia elongata) Associated with Plant Immunity to Biotic and Abiotic Stress Factors.

Plant defense polypeptides play a crucial role in providing plants with constitutive immunity against various biotic and abiotic stressors. In this study, we explored a complex of proteins from wheatgrass (Elytrigia elongata) spikelets to estimate their role in the plant's tolerance to various environmental factors. The current research shows that in vitro protein extracts from E. elongata spikelets possess antifungal activity against certain Fusarium species, which are specific cereal pathogens, at concentrations of 1-2 mg/mL. In this study, we reproduced these antifungal activities using a 4 mg/mL extract in artificial fungal infection experiments on wheat grain (Triticum aestivum) under controlled laboratory conditions. Furthermore, the tested extract demonstrated a protective effect on Saccharomyces cerevisiae exposed to hyper-salinity stress at a concentration of 2 mg/mL. A combined scheme of fractionation and structural identification was applied for the estimation of the diversity of defense polypeptides. Defensins, lipid-transfer proteins, hydrolase inhibitors (cereal bifunctional trypsin/alpha-amylase inhibitors from a Bowman-Birk trypsin inhibitor), and high-molecular-weight disease resistance proteins were isolated from the extract. Thus, wheatgrass spikelets appear to be a reservoir of defense polypeptides. Our findings contribute to a deeper understanding of plant defense proteins and peptides and their involvement in the adaptation to various stress factors, and they reveal the regulatory effect at the ecosystem level.

BnUC1 Is a Key Regulator of Epidermal Wax Biosynthesis and Lipid Transport in Brassica napus.

The bHLH (basic helix-loop-helix) transcription factor AtCFLAP2 regulates epidermal wax accumulation, but the underlying molecular mechanism remains unknown. We obtained BnUC1mut (BnaA05g18250D homologous to AtCFLAP2) from a Brassica napus mutant with up-curling leaves (Bnuc1) and epidermal wax deficiency via map-based cloning. BnUC1mut contains a point mutation (N200S) in the conserved dimerization domain. Overexpressing BnUC1mut in ZS11 (Zhongshuang11) significantly decreased the leaf epidermal wax content, resulting in up-curled and glossy leaves. In contrast, knocking out BnUC1mut in ZS11-NIL (Zhongshuang11-near-isogenic line) restored the normal leaf phenotype (i.e., flat) and significantly increased the leaf epidermal wax content. The point mutation weakens the ability of BnUC1mut to bind to the promoters of VLCFA (very-long-chain fatty acids) synthesis-related genes, including KCS (β-ketoacyl coenzyme synthase) and LACS (long-chain acyl CoA synthetase), as well as lipid transport-related genes, including LTP (non-specific lipid transfer protein). The resulting sharp decrease in the transcription of genes affecting VLCFA biosynthesis and lipid transport disrupts the normal accumulation of leaf epidermal wax. Thus, BnUC1 influences epidermal wax formation by regulating the expression of LTP and genes associated with VLCFA biosynthesis. Our findings provide a foundation for future investigations on the mechanism mediating plant epidermal wax accumulation.

Glycosylphosphatidylinositol-anchored lipid transfer proteins influence root cap cuticle formation at primary root tips, promoting NaCl tolerance in Arabidopsis seedlings.

Root cap cuticles (RCCs), comprising mainly very-long-chain fatty acids (VLCFAs), promote salt tolerance by preventing ion influx. Glycosylphosphatidylinositol-anchored lipid transfer protein (LTPG)1 and LTPG2 participate in VLCFA deposition in the extracellular region, aiding RCC formation in the lateral roots. In this study, we investigated whether LTPG1 and LTPG2 have similar functions in the primary roots of young Arabidopsis thaliana. Phenotypic analyses, fluorescence microscopy, and quantitative real-time reverse transcription polymerase chain reaction confirmed that NaCl exposure induced LTPG1 and LTPG2 expression and promoted RCC formation in young primary roots. The loss of RCC in the ltpg1 and ltpg2 mutants resulted in increased NaCl sensitivity of root elongation. NaCl also upregulated the expression of several NaCl-responsive genes in ltpg1 and ltpg2. We conclude that RCC formation via LTPG function is pivotal in enhancing salt tolerance in young primary roots.

Immunolocalization of hordein synthesis and transport in developing barley endosperm.

The spatial accumulation of hordeins in the developing endosperm of barley grains was examined by immunofluorescence microscopy (immunolight microscopy [iLM]) and immunoelectron microscopy (iEM) to establish the timing and subcellular pattern of hordein synthesis and deposition. The pattern seen for hordeins was compared to other abundant grain proteins, such as serpin Z4 and lipid transfer protein 1 (LTP1). Hordein accumulates throughout grain development, from 6 to 37 days post-anthesis (DPA). In contrast, serpin Z4 was present at 6 DPA, but the greatest synthesis and accumulation occurred during the middle of seed development, from 15 to 30 DPA. LTP1 accumulated later in seed development, from 15 to 30 DPA. Hordeins accumulated within the lumen of the endoplasmic reticulum (ER), were exocytosed from the ER membrane, and accumulated in protein bodies, which then fused either with the protein storage vacuoles or with other protein bodies, which also later fused with the protein storage vacuoles. iEM showed hordein, and LTP1 appeared not to traverse the Golgi apparatus (GA). Hordein, LTP1, and serpin Z4 colocalized to the same protein bodies and were co-transported to the protein storage vacuole in the same protein bodies. It is likely that this represents a general transport mechanism common to storage proteins in developing grains.

Long-chain fatty acids block allergic reaction against lipid transfer protein Sola l 7 from tomato seeds.

Due to the benefits of tomato as an antioxidant and vitamin source, allergy to this vegetable food is a clinically concerning problem. Sola l 7, a class I lipid transfer protein found in tomato seeds, has been identified as an allergen linked to severe anaphylaxis. However, the role of lipid binding in Sola l 7-induced allergy remains unclear. Here, the three-dimensional structure of recombinant Sola l 7 (rSola l 7) has been elucidated using nuclear magnetic resonance spectroscopy (NMR). Its interaction with free fatty acids has been deeply studied; fluorescence emission spectroscopy revealed that different long-chain fatty acids interact with the protein, affecting the only tyrosine residue present in Sola l 7. On the contrary, no changes in the overall secondary structure were observed after the analysis of the circular dichroism spectra in the presence of fatty acids. Unsaturated oleic and linoleic fatty acids presented higher affinity and promoted more significant changes than saturated or short-chain fatty acids. 1H-15N HSQC NMR spectra allowed to determine the regions of the protein that were modified when rSola l 7 interacts with the fatty acids, suggesting epitope modification after the interaction. For corroboration, IgG and IgE binding to rSola l 7 were assessed in the presence of free fatty acids, revealing that both IgE and IgG binding were significantly lower than in their absence, suggesting a potential protective role of unsaturated fatty acids in tomato allergy.

The Diagnosis of Allergy to Lipid Transfer Proteins.

To provide an update on the diagnosis of non-specific Lipid Transfer Protein (nsLTP) allergy. More publications report the presence of nsLTP allergy in Northern European countries and nsLTP sensitisation in children. Individuals are more likely to have severe reactions if there is recognition of increasing numbers of LTP components. Diagnosis is problematic; not all those with nsLTP allergy will have a positive test to a peach extract containing Pru p 3, the peach nsLTP. Sensitisation to nsLTP is being reported in more countries, including to the nsLTP in Cannabis Sativa in North America. Meals containing multiple nsLTP foods are more likely to be involved in co-factor reactions. Component-resolved diagnostics are superior to skin prick tests, to determine sensitisation to the individual nsLTP allergens causing symptoms and, in the future, the Basophil Activation test may best discriminate between sensitization and clinical allergy.

Possibility of Using a Composition with Lipid Transfer Protein Ns-LTP1 in Therapy of Ulcerative Colitis

Possibility of Using a Composition with Lipid Transfer Protein Ns-LTP1 in Therapy of Ulcerative Colitis

SlLTPg1, a tomato lipid transfer protein, positively regulates in response to biotic stresses

Late blight, caused by Phytophthora infestans (P. infestans), is among the most devastating diseases affecting tomato and other Solanaceae species. Lipid transfer proteins (LTPs) represent a class of small, basic proteins that play a crucial role in combating biotic stresses. Previous studies have shown that SlLTPg1 most strongly responds after P. infestans infestation among the LTPs family in tomato. However, the function of SlLTPg1 in disease resistance remains unclear. Here, we constructed transient overexpression and VIGS-silenced plants of SlLTPg1. Our results revealed that SlLTPg1 plays a regulatory role in enhancing tomato resistance against P. infestans. This enhancement was attributed to the upregulation of defense-related genes and reactive oxygen species (ROS) scavenging genes, as well as increased enzymatic antioxidant activities. Importantly, we found that the SlLTPg1 protein significantly inhibited the growth of Fusarium oxysporum (F. oxysporum) by observing the zone of inhibition. Interestingly, we found smaller lesion diameters and upregulated expression levels of PR genes in transient overexpression SlLTPg1 of tobacco. Therefore, we further constructed transgenic tobacco lines of SlLTPg1, presenting evidence that overexpression of SlLTPg1 could positively regulate the resistance of tobacco to F. oxysporum. These findings revealed the role of SlLTPg1 in tomato resistance to P. infestans and tobacco resistance to F. oxysporum. Moreover, we propose SlLTPg1 as a potential candidate gene for augmenting broad-spectrum plant resistance against pathogens.

Whole-genome sequencing and identification of antimicrobial peptide coding genes in parsley (Petroselinum crispum), an important culinary and medicinal Apiaceae species.

Parsley is a commonly cultivated Apiaceae species of culinary and medicinal importance. Parsley has several recognized health benefits and the species has been utilized in traditional medicine since ancient times. Although parsley is among the most commonly cultivated members of Apiaceae, no systematic genomic research has been conducted on parsley. In the present work, parsley genome was sequenced using the long-read HiFi (high fidelity) sequencing technology and a draft contig assembly of 1.57 Gb that represents 80.9% of the estimated genome size was produced. The assembly was highly repeat-rich with a repetitive DNA content of 81%. The assembly was phased into a primary and alternate assembly in order to minimize redundant contigs. Scaffolds were constructed with the primary assembly contigs, which were used for the identification of AMP (antimicrobial peptide) genes. Characteristic AMP domains and 3D structures were used to detect and verify antimicrobial peptides. As a result, 23 genes (PcAMP1-23) representing defensin, snakin, thionin, lipid transfer protein and vicilin-like AMP classes were identified. Bioinformatic analyses for the characterization of peptide physicochemical properties indicated that parsley AMPs are extracellular peptides, therefore, plausibly exert their antimicrobial effects through the most commonly described AMP action mechanism of membrane attack. AMPs are attracting increasing attention since they display their fast antimicrobial effects in small doses on both plant and animal pathogens with a significantly reduced risk of resistance development. Therefore, identification and characterization of AMPs is important for their incorporation into plant disease management protocols as well as medicinal research for the treatment of multi-drug resistant infections.

Pollen-Food Allergy Syndrome: From Food Avoidance to Deciphering the Potential Cross-Reactivity between Pru p 3 and Ole e 7.

Cross-reactivity between nonspecific lipid transfer proteins could cause anaphylaxis, further influencing food avoidance and nutrient deficiencies. The one affecting olive pollen (Ole e 7) and peach (Pru p 3) may underlie a variety of pollen-food syndromes, though a deep molecular analysis is necessary. Three Ole e 7-monosensitised patients (MON_OLE), three Pru p 3-monosensitised patients (MON_PRU) and three bisensitised patients (BI) were selected. For epitope mapping, both digested proteins were incubated with patient sera, and the captured IgE-bound peptides were characterised by LC-MS. The analysis revealed two Ole e 7 epitopes and the three Pru p 3 epitopes previously described. Interestingly, the "KSALALVGNKV" Ole e 7 peptide was recognised by MON_OLE, BI and MON_PRU patients. Conversely, all patients recognised the "ISASTNCATVK" Pru p 3 peptide. Although complete sequence alignment between both proteins revealed 32.6% identity, local alignment considering seven residue fragments showed 50 and 57% identity when comparing "ISASTNCATVK" with Ole e 7 and "KSALALVGNKV" with Pru p 3. This study mapped sIgE-Ole e 7-binding epitopes, paving the way for more precise diagnostic tools. Assuming non-significant sequence similarity, structural homology and shared key residues may underlie the potential cross-reactivity between Ole e 7 and Pru p 3 nsLTPs.