Combined Effects of Pressure and Ionic Strength on Protein-Protein Interactions: An Empirical Approach.

Proteins are exposed to hydrostatic pressure (HP) in a variety of ecosystems as well as in processing steps such as freeze-thaw, cell disruption, sterilization, and homogenization, yet pressure effects on protein-protein interactions (PPIs) remain underexplored. With the goal of contributing toward the expanded use of HP as a fundamental control parameter in protein research, processing, and engineering, small-angle X-ray scattering was used to examine the effects of HP and ionic strength on ovalbumin, a model protein. Based on an extensive data set, we develop an empirical method for scaling PPIs to a master curve by combining HP and osmotic effects. We define an effective pressure parameter that has been shown to successfully apply to other model protein data available in the literature, with deviations evident for proteins that do not follow the apparent Hofmeister series. The limitations of the empirical scaling are discussed in the context of the hypothesized underlying mechanisms.

Neutron Scattering Analysis of Cryptococcus neoformans Polysaccharide Reveals Solution Rigidity and Repeating Fractal-like Structural Patterns.

Cryptococcus neoformans is a fungal pathogen that can cause life-threatening brain infections in immunocompromised individuals. Unlike other fungal pathogens, it possesses a protective polysaccharide capsule that is crucial for its virulence. During infections, Cryptococcus cells release copious amounts of extracellular polysaccharides (exo-PS) that interfere with host immune responses. Both exo-PS and capsular-PS play pivotal roles in Cryptococcus infections and serve as essential targets for disease diagnosis and vaccine development strategies. However, understanding their structure is complicated by their polydispersity, complexity, sensitivity to sample isolation and processing, and scarcity of methods capable of isolating and analyzing them while preserving their native structure. In this study, we employ small-angle neutron scattering (SANS) and ultra-small-angle neutron scattering (USANS) for the first time to investigate both fungal cell suspensions and extracellular polysaccharides in solution. Our data suggests that exo-PS in solution exhibits collapsed chain-like behavior and demonstrates mass fractal properties that indicate a relatively condensed pore structure in aqueous environments. This observation is also supported by scanning electron microscopy (SEM). The local structure of the polysaccharide is characterized as a rigid rod, with a length scale corresponding to 3-4 repeating units. This research not only unveils insights into exo-PS and capsular-PS structures but also demonstrates the potential of USANS for studying changes in cell dimensions and the promise of contrast variation in future neutron scattering studies.

Drug-Dependent Morphological Transitions in Spherical and Worm-Like Polymeric Micelles Define Stability and Pharmacological Performance of Micellar Drugs.

Significant advances in physicochemical properties of polymeric micelles enable optimization of therapeutic drug efficacy, supporting nanomedicine manufacturing and clinical translation. Yet, the effect of micelle morphology on pharmacological efficacy is not adequately addressed. This work addresses this gap by assessing pharmacological efficacy of polymeric micelles with spherical and worm-like morphologies. It is observed that poly(2-oxazoline)-based polymeric micelles can be elongated over time from a spherical structure to worm-like structure, with elongation influenced by several conditions, including the amount and type of drug loaded into the micelles. The role of different morphologies on pharmacological performance of drug loaded micelles against triple-negative breast cancer and pancreatic cancer tumor models is further evaluated. Spherical micelles accumulate rapidly in the tumor tissue while retaining large amounts of drug; worm-like micelles accumulate more slowly and only upon releasing significant amounts of drug. These findings suggest that the dynamic character of the drug-micelle structure and the micelle morphology play a critical role in pharmacological performance, and that spherical micelles are better suited for systemic delivery of anticancer drugs to tumors when drugs are loosely associated with the polymeric micelles.

In Situ Monitoring of Protein Unfolding/Structural States under Cold High-Pressure Stress.

Biopharmaceutical formulations may be compromised by freezing, which has been attributed to protein conformational changes at a low temperature, and adsorption to ice-liquid interfaces. However, direct measurements of unfolding/conformational changes in sub-0 °C environments are limited because at ambient pressure, freezing of water can occur, which limits the applicability of otherwise commonly used analytical techniques without specifically tailored instrumentation. In this report, small-angle neutron scattering (SANS) and intrinsic fluorescence (FL) were used to provide in situ analysis of protein tertiary structure/folding at temperatures as low as -15 °C utilizing a high-pressure (HP) environment (up to 3 kbar) that prevents water from freezing. The results show that the α-chymotrypsinogen A (aCgn) structure is reasonably maintained under acidic pH (and corresponding pD) for all conditions of pressure and temperature tested. On the other hand, reversible structural changes and formation of oligomeric species were detected near -10 °C via HP-SANS for ovalbumin under neutral pD conditions. This was found to be related to the proximity of the temperature of cold denaturation of ovalbumin (TCD ∼ -17 °C; calculated via isothermal chemical denaturation and Gibbs-Helmholtz extrapolation) rather than a pressure effect. Significant structural changes were also observed for a monoclonal antibody, anti-streptavidin IgG1 (AS-IgG1), under acidic conditions near -5 °C and a pressure of ∼2 kbar. The conformational perturbation detected for AS-IgG1 is proposed to be consistent with the formation of unfolding intermediates such as molten globule states. Overall, the in situ approaches described here offer a means to characterize the conformational stability of biopharmaceuticals and proteins more generally under cold-temperature stress by the assessment of structural alteration, self-association, and reversibility of each process. This offers an alternative to current ex situ methods that are based on higher temperatures and subsequent extrapolation of the data and interpretations to the cold-temperature regime.

l-Arginine supplementation of gilts during early gestation modulates energy sensitive pathways in pig conceptuses.

Dietary l-arginine (ARG) supplementation has been studied as a nutritional strategy to improve reproductive performance of pregnant sows, since arginine is a conditionally essential amino acid. However, reports addressing the molecular mechanisms that mediate supplementation effects on embryos and fetuses development are still scarce. Therefore, we aimed to evaluate the effects of 1.0% ARG supplementation of commercial pregnant gilts on genes and proteins from energy metabolism and antioxidant defense pathways in embryos and fetuses. We also analyzed the global transcriptome profile of 25- and 35-day-old conceptuses. At Day 25, we observed a lower abundance of phospho-AMP-activated protein kinase (phospho-AMPK) protein and downregulation of oxidative phosphorylation system genes in ARG embryos. On the other hand, ARG fetuses showed greater expression of MLST8 and lower expression of MTOR genes, in addition to lower abundance of phospho-AMPK and phospho-mammalian target of rapamycin (phospho-mTOR) proteins. Transcriptome analysis at Day 35 did not present differentially expressed genes. For the antioxidant defense pathway, no differences were found between CON and ARG conceptuses, only trends. In general, supplementation of gilts with 1.0% ARG during early gestation affects energy sensitive pathways in 25- and 35-day conceptuses; however, no effects of supplementation were found on the antioxidative defense pathway in 25-day embryos.

Effect of Phosphorylation on a Human-like Osteopontin Peptide.

The last decade established that the dynamic properties of the phosphoproteome are central to function and its modulation. The temporal dimension of phosphorylation effects remains nonetheless poorly understood, particularly for intrinsically disordered proteins. Osteopontin, selected for this study due to its key role in biomineralization, is expressed in many species and tissues to play a range of distinct roles. A notable property of highly phosphorylated isoforms of osteopontin is their ability to sequester nanoclusters of calcium phosphate to form a core-shell structure, in a fluid that is supersaturated but stable. In Biology, this process enables soft and hard tissues to coexist in the same organism with relative ease. Here, we extend our understanding of the effect of phosphorylation on a disordered protein, the recombinant human-like osteopontin rOPN. The solution structures of the phosphorylated and unphosphorylated rOPN were investigated by small-angle x-ray scattering and no significant changes were detected on the radius of gyration or maximum interatomic distance. The picosecond-to-nanosecond dynamics of the hydrated powders of the two rOPN forms were further compared by elastic and quasi-elastic incoherent neutron scattering. Phosphorylation was found to block some nanosecond side-chain motions while increasing the flexibility of other side chains on the faster timescale. Phosphorylation can thus selectively change the dynamic behavior of even a highly disordered protein such as osteopontin. Through such an effect on rOPN, phosphorylation can direct allosteric mechanisms, interactions with substrates, cofactors and, in this case, amorphous or crystalline biominerals.

Structural studies of hydrated samples of amorphous calcium phosphate and phosphoprotein nanoclusters.

There are abundant examples of nanoclusters and inorganic microcrystals in biology. Their study under physiologically relevant conditions remains challenging due to their heterogeneity, instability, and the requirements of sample preparation. Advantages of using neutron diffraction and contrast matching to characterize biomaterials are highlighted in this article. We have applied these and complementary techniques to search for nanocrystals within clusters of calcium phosphate sequestered by bovine phosphopeptides, derived from osteopontin or casein. The neutron diffraction patterns show broad features that could be consistent with hexagonal hydroxyapatite crystallites smaller than 18.9 Å. Such nanocrystallites are, however, undetected by the complementary X-ray and FTIR data, collected on the same samples. The absence of a distinct diffraction pattern from the nanoclusters supports the generally accepted amorphous calcium phosphate structure of the mineral core.

Mineralisation of soft and hard tissues and the stability of biofluids.

Evidence is provided from studies on natural and artificial biofluids that the sequestration of amorphous calcium phosphate by peptides or proteins to form nanocluster complexes is of general importance in the control of physiological calcification. A naturally occurring mixture of osteopontin peptides was shown, by light and neutron scattering, to form calcium phosphate nanoclusters with a core-shell structure. In blood serum and stimulated saliva, an invariant calcium phosphate ion activity product was found which corresponds closely in form and magnitude to the ion activity product observed in solutions of these osteopontin nanoclusters. This suggests that types of nanocluster complexes are present in these biofluids as well as in milk. Precipitation of amorphous calcium phosphate from artificial blood serum, urine and saliva was determined as a function of pH and the concentration of osteopontin or casein phosphopeptides. The position of the boundary between stability and precipitation was found to agree quantitatively with the theory of nanocluster formation. Artificial biofluids were prepared that closely matched their natural counterparts in calcium and phosphate concentrations, pH, saturation, ionic strength and osmolality. Such fluids, stabilised by a low concentration of sequestering phosphopeptides, were found to be highly stable and may have a number of beneficial applications in medicine.

Intraocular Pressure Values using IcareⓇ Rebound Tonometer and Correlation with Postconceptional Age in Premature Infants.

Purpose: This study aimedto determine a normative range of intraocular pressure (IOP) values measured with Icare rebound tonometer in premature infants and evaluate IOP variation over time and its correlation with the progression of postconceptional age (PCA). By doing so, we also evaluated advantages of this IOP-measuring method in this population when compared to more traditional methods. Methods: We conducted a single-center prospective study that included premature infants (gestational age ≤32 weeks) who were admitted to the neonatal intensive care unit (NICU) in Hospital Professor Doutor Fernando Fonseca. The study took place between January and December 2021. IOP was measured using Icare tonometer on the occasion of the first retinopathy of prematurity (ROP) screening requested by the NICU and again after a two-week interval if PCA was still ≤37 weeks. IOP measurements were stopped at 37 weeks or if the infant was discharged. The evaluated outcomes were mean IOP values and their correlation with PCA. Results: Thirty-four eyes of 17 preterm infants with a mean gestational age of 29.4 ± 2.3 weeks and a mean birth weight of 1222.9 ± 361.9 gr were evaluated. The mean IOP registered was 16.1 ± 6.4 mmHg, with a median value of 15.3 mmHg. The top 90th percentile was 22.1 mmHg and the bottom 10th percentile was 9.0 mmHg. The average IOP reduction was 4.8 ± 6.7 mmHg (P = 0.0019) within the two-week interval of PCA. Conclusion: The mean IOP in premature infants was 16.1 ± 6.4 mmHg and this value significantly decreased by 4.8 ± 6.7 mmHg every two weeks of PCA.

High Pressure Light Scattering of Therapeutic Proteins To Probe Aggregation and Protein-Protein Interactions.

There is interest in the direct in situ measurement of protein aggregation and reversible protein-protein interactions at high pressure as a means to assess protein stability. This is currently limited by the availability of in-house analytical methods. High-pressure (HP) scattering instrumentation (using either neutrons, X-rays, or light sources) are relatively rare, due to extensive development hurdles and lack of standardization. This report focuses on design, operation, and application of a new HP light scattering apparatus based on commercially available equipment with a view to wider applications. HP static light scattering results were obtained for two monoclonal antibodies (MAbs) that exhibit different extents of unfolding and aggregation at these conditions. Aggregation that was observed during in situ pressure incubations varied by MAb and total ionic strength of solution. This was conducted in tandem with ex situ measurements on MAb solutions that were incubated under pressure, where monomer loss was measured with size exclusion chromatography. Pressure cycling was also used to assess the extent of pressure-induced reversible and irreversible aggregation. Finally, the ability of the HP light scattering apparatus to assess the influence of pressure on reversible protein-protein interactions in the canonical sense of second osmotic virial coefficients was assessed using lysozyme, a relatively well-characterized protein under hydrostatic pressure. The method offers a convenient and reproducible capability that complements current small angle neutron/X-ray instrumentation, providing measurements that can be used to optimize the planning and interpretation of scattering data from synchrotron or neutron research facilities. Our results address a growing demand to characterize protein aggregates and aggregation-prone partially unfolded intermediates.

Neutron Scattering Analysis of Cryptococcus neoformans Polysaccharide Reveals Solution Rigidity and Repeating Fractal-like Structural Patterns.

Cryptococcus neoformans is a fungal pathogen that can cause life-threatening brain infections in immunocompromised individuals. Unlike other fungal pathogens, it possesses a protective polysaccharide capsule that is crucial for its virulence. During infections, Cryptococcus cells release copious amounts of extracellular polysaccharides (exo-PS) that interfere with host immune responses. Both exo-PS and capsular-PS play pivotal roles in Cryptococcus infections and serve as essential targets for disease diagnosis and vaccine development strategies. However, understanding their structure is complicated by their polydispersity, complexity, sensitivity to sample isolation and processing, and scarcity of methods capable of isolating and analyzing them while preserving their native structure. In this study, we employ small-angle neutron scattering (SANS) and ultra-small angle neutron scattering (USANS) for the first time to investigate both fungal cell suspensions and extracellular polysaccharides in solution. Our data suggests that exo-PS in solution exhibits collapsed chain-like behavior and demonstrates mass fractal properties that indicate a relatively condensed pore structure in aqueous environments. This observation is also supported by scanning electron microscopy (SEM). The local structure of the polysaccharide is characterized as a rigid rod, with a length-scale corresponding to 3 to 4 repeating units. This research not only unveils insights into exo-PS and capsular-PS structures but also demonstrates the potential of USANS for studying changes in cell dimensions and the promise of contrast variation in future neutron scattering studies.

Isotretinoin-Induced Distal Ileitis Mimicking Crohn's Disease.

Terminal ileitis is a common condition defined as inflammation of the terminal portion of the ileum, which is typically associated with inflammatory bowel disease (IBD), classically Crohn's disease (CD). However, it can have other etiologies, including drug-induced ones. Isotretinoin is an effective and commonly used treatment for acne vulgaris, presenting multiple adverse effects. There have been discussions over its association with enteric inflammation, particularly over IBD emergence risk. We report a case of a previously healthy 17-year-old female who presented transitory clinical, laboratory, imaging, and endoscopic evidence of distal ileitis, temporally related to extended isotretinoin treatment and mimicking CD. Repeated clinical, laboratory, imaging, and endoscopic reassessment after isotretinoin discontinuation confirmed an almost complete resolution of the condition, avoiding IBD misdiagnosis and specific medication initiation. Our case highlights the differential diagnosis of ileitis as being of critical importance to avoid further unnecessary diagnostic investigations and inadequate treatment. Serial re-evaluation may be of key importance to reach a final diagnosis. Although recent literature suggests that isotretinoin is not associated with an increased IBD risk, our case highlights the possibility of it inducing small bowel injury and inflammation, similar to what has been reported with other drugs.

Late versus prophylactic chorioretinectomy for the prevention of trauma-related proliferative vitreoretinopathy.

INTRODUCTION: Certain injuries, especially those with a deep-impact (involving the choroid and even the sclera) intraocular foreign body (IOFB), have a high risk for developing either full-blown proliferative vitreoretinopathy (PVR) or full-thickness retinal folds. Although less severe than the former, this so-called 'stage 0 PVR' can severely impact vision, and effective treatment for these folds has not existed heretofore. PATIENTS AND METHODS: Four eyes of 4 patients sustained an IOFB injury with deep impact. All eyes underwent vitrectomy and IOFB removal soon after the injury, and all eyes showed substantial visual improvement postoperatively. However, in a few months the visual acuity dropped again, due to the development of full-thickness retinal folds radiating from the scar. All four eyes then underwent a second vitrectomy with (late rather than prophylactic) chorioretinectomy by creating a 1-mm-wide ring of bare sclera around the scar. The highest setting of the diathermy machine was used as the endodiathermy probe evaporated both the retina and the choroid to create the ring. Laser retinopexy to surround the ring was used only if the lesion was not in the posterior pole. RESULTS: Within a few days, the retinal folds completely disappeared in each eye, and the visual acuity reached the highest earlier value seen after the initial surgery. All patients have long-term follow-up (mean, 22 months) with no postoperative complications. CONCLUSIONS: Chorioretinectomy, although it is ideally used as a prophylaxis against PVR formation and the development of retinal fold formation, also proved equally effective as a late treatment option in the presence of such folds. Such late chorioretinectomy, however, is applicable only for eyes with deep-impact IOFB injuries, not for eyes with a perforating injury or rupture.

Incidence and Risk Factors for Retinopathy of Prematurity in a Portuguese Cohort.

PURPOSE: To evaluate the incidence and risk factors for retinopathy of prematurity (ROP) in two Portuguese neonatal units with a sub-analysis of infants with a gestational age (GA) of 28 weeks or older. METHODS: This was a retrospective case series of all infants who underwent ROP screening from 2012 to 2020. Demographic, clinical, and laboratory data were collected. Univariate logistic regression was used to examine the risk factors for ROP followed by multivariate regression. RESULTS: A total of 475 infants were included with a median GA of 30 weeks (range: 23 to 36 weeks) and a median birth weight of 1,229 grams (range: 408 to 2,620 grams). ROP was diagnosed in 113 infants (23.8%) and 29 (6.1%) were treated. In the multivariate analysis, GA and hyperglycemia were significantly associated with severe ROP (P < .001). In the subgroup analysis of infants with a GA of 28 weeks or older, bronchopulmonary dysplasia, late-onset sepsis, and hyperglycemia were linked to severe ROP. CONCLUSIONS: The incidence of ROP in the cohort falls within the range of other high-income countries. Hyperglycemia overpowered all of the other risk factors. Although rare, more mature infants are also at risk for severe ROP. Infants with older GA share the same group of risk factors, but bronchopulmonary dysplasia seems to play a greater role. [J Pediatr Ophthalmol Strabismus. 2022;59(4):254-260.].

Temporal macular thinning and vessel density correlation in children and young adults with sickle cell disease.

Optical coherence tomography angiography (OCTA) is a recent noninvasive imaging technology that has proved to provide a comprehensive evaluation of retinal vascular abnormalities in adult patients with sickle cell disease (SCD). However, the pediatric population remains less studied. The purpose of this study is to evaluate the correlation between temporal vessel density (VD) in OCTA and temporal macular thinning in optical coherence tomography (OCT) in children and young adults with SCD. We reviewed medical records, OCT and OCTA (Spectralis®) scans from a sample of 32 eyes from 16 patients (7 female + 9 male) under 25 years old. The OCT macular thickness data and OCTA image data were processed using Python 3.9 programming language, and statistical analysis was performed. Pearson Correlation Coefficient between macular thickness and VD for inner and outer temporal areas was 0.47 (p-value = 0.006) and 0.74 (p-value < 0.001), respectively. A t-test was also performed to prove that there is a statistically significant VD difference in patients with higher and lower macular thickness (p < 0.001). These results show that children with outer temporal macular thinning on OCT have lower outer temporal VD on OCTA suggesting that microvascular insults may lead to chronic ischemic changes in the inner retinal layers.

Retrospective comparison between growth and retinopathy of prematurity model versus WINROP model.

OBJECTIVE: To compare the weight and insulin-like growth factor-1 in neonatal retinopathy (WINROP) to the growth and retinopathy of prematurity (G-ROP) model in a Portuguese cohort. DESIGN: Retrospective case series. METHODS: Clinical records of consecutive infants who underwent retinopathy of prematurity (ROP) screening from April 2012 to May 2019 were retrospectively reviewed. Both WINROP and G-ROP models were accessed for sensitivity and specificity for type 1 ROP. A separate analysis of both algorithms was performed in infants with gestational age (GA) <30 weeks. RESULTS: Of the 375 infants included in the study, 313 were eligible for G-ROP analysis and 311 for WINROP. In the G-ROP group, 22 infants developed type 1 ROP (sensitivity 90.91%, 95% confidence interval [CI] 70.84%-98.98%). In the WINROP group, 23 infants needed treatment (sensitivity of 86.96%, 95% CI 66.41%-97.22%). Both models reached 100% sensitivity for type 1 ROP if restricted to GA <30 weeks. CONCLUSIONS: Both models were easy to use and had similar sensitivities. If restricted to GA <30 weeks, both models detected all type 1 ROP.

High-Pressure, Low-Temperature Induced Unfolding and Aggregation of Monoclonal Antibodies: Role of the Fc and Fab Fragments.

The effects of high pressure and low temperature on the stability of two different monoclonal antibodies (MAbs) were examined in this work. Fluorescence and small-angle neutron scattering were used to monitor the in situ effects of pressure to infer shifts in tertiary structure and characterize aggregation prone intermediates. Partial unfolding was observed for both MAbs, to different extents, under a range of pressure/temperature conditions. Fourier transform infrared spectroscopy was also used to monitor ex situ changes in secondary structure. Preservation of native secondary structure after incubation at elevated pressures and subzero ° C temperatures was independent of the extent of tertiary unfolding and reversibility. Several combinations of pressure and temperature were also used to discern the respective contributions of the isolated Ab fragments (Fab and Fc) to unfolding and aggregation. The fragments for each antibody showed significantly different partial unfolding profiles and reversibility. There was not a simple correlation between stability of the full MAb and either the Fc or Fab fragment stabilities across all cases, demonstrating a complex relationship to full MAb unfolding and aggregation behavior. That notwithstanding, the combined use of spectroscopic and scattering techniques provides insights into MAb conformational stability and hysteresis in high-pressure, low-temperature environments.

DIGIROP efficacy for detecting treatment-requiring retinopathy of prematurity in a Portuguese cohort.

BACKGROUND/OBJECTIVES: To determine the efficacy of the DIGIROP model in detecting treatment-requiring retinopathy of prematurity (TR-ROP) in a Portuguese cohort. SUBJECTS/METHODS: Multicentre, retrospective cohort study of all consecutive preterm infants who underwent ROP screening from April 2012 to May 2019 in two neonatal units. Gestational age (GA), birth weight (BW) and sex were inserted in the DIGIROP platform. The optimal cut-off point to achieve 100% sensitivity was calculated. Area under the receiver operating characteristic curve (AUC) was calculated. RESULTS: Of the 431 infants who underwent ROP screening, 257 were eligible for DIGIROP analysis and 174 infants were excluded for having a GA outside the range 24-30 weeks imposed by the DIGIROP algorithm. Median GA was 29 weeks (range 24-30) and BW was 1060 g (range 408-2080). Twenty-tree infants (8.9%) developed TR-ROP. The highest risk obtained for TR-ROP was 0.5404 (95% CI 0.4343-0.6616) with a median achieved risk of 0.0938 (range 0.0016-0.5404). The optimal cut-off point to achieve 100% sensitivity on TR-ROP was 0.0016. The number of infants receiving ROP examinations would have been reduced from 257 to 187 infants (-27.2%) if the model was applied. CONCLUSIONS: In our cohort, of 257 infants, the optimal cut-off point to achieve 100% sensitivity for TR-ROP was 0.0016 with moderate accuracy in the AUC (0.70). The number of infants requiring screening would have decreased 27.2% if the model was applied. It is essential that algorithms continue to be tested in different populations, especially in cohorts that include both younger and older GA infants.