The elementary reactions for incorporation into crystals.

The growth rates of crystals are largely dictated by the chemical reaction between solute and kinks, in which a solute molecule severs its bonds with the solvent and establishes new bonds with the kink. Details on this sequence of bond breaking and rebuilding remain poorly understood. To elucidate the reaction at the kinks we employ four solvents with distinct functionalities as reporters on the microscopic structures and their dynamics along the pathway into a kink. We combine time-resolved in situ atomic force microscopy and x-ray and optical methods with molecular dynamics simulations. We demonstrate that in all four solvents the solute, etioporphyrin I, molecules reach the steps directly from the solution; this finding identifies the measured rate constant for step growth as the rate constant of the reaction between a solute molecule and a kink. We show that the binding of a solute molecule to a kink divides into two elementary reactions. First, the incoming solute molecule sheds a fraction of its solvent shell and attaches to molecules from the kink by bonds distinct from those in its fully incorporated state. In the second step, the solute breaks these initial bonds and relocates to the kink. The strength of the preliminary bonds with the kink determines the free energy barrier for incorporation into a kink. The presence of an intermediate state, whose stability is controlled by solvents and additives, may illuminate how minor solution components guide the construction of elaborate crystal architectures in nature and the search for solution compositions that suppress undesirable or accelerate favored crystallization in industry.

Particle dispersion through porous media with heterogeneous attractions.

Porous media used in many practical applications contain natural spatial variations in composition and surface charge that lead to heterogeneous physicochemical attractions between the media and transported particles. We performed Stokesian dynamics (SD) simulations to examine the effects of heterogeneous attractions on quiescent diffusion and hydrodynamic dispersion of particles within geometrically ordered arrays of nanoposts. We find that transport under quiescent conditions occurs by two mechanisms, diffusion through the void space and intermittent hopping between the attractive wells of different nanoposts. As the attraction heterogeneity increases, the latter mechanism becomes dominant, resulting in an increase in the particle trajectory tortuosity, deviations from Gaussian behavior in the particle displacement distributions, and a decrease in the long-time particle diffusivity. Similarly, under flow conditions corresponding to low Péclet number (Pe), increased attraction heterogeneity leads to transient localization near the nanoposts, resulting in a broadening of the particle distribution and enhanced longitudinal dispersion in the direction of flow. At high Pe where advection strongly dominates, however, the longitudinal dispersion coefficient is insensitive to attraction heterogeneity and exhibits Taylor-Aris dispersion behavior. Our findings provide insight into how heterogeneous interactions may influence particle transport in complex 3-D porous media.

Confined Dynamics in Spherical Polymer Brushes.

We investigate the dynamics of polymers grafted to spherical nanoparticles in solution using hybrid molecular dynamics simulations with a coarse-grained solvent modeled via the multiparticle collision dynamics algorithm. The mean-square displacements of monomers near the surface of the nanoparticle exhibit a plateau on intermediate time scales, indicating confined dynamics reminiscent of those reported in neutron spin-echo experiments. The confined dynamics vanish beyond a specific radial distance from the nanoparticle surface that depends on the polymer grafting density. We show that this dynamical confinement transition follows theoretical predictions for the critical distance associated with the structural transition from confined to semidilute brush regimes. These findings suggest the existence of a hitherto unreported dynamic length scale connected with theoretically predicted static fluctuations in spherical polymer brushes and provide new insights into recent experimental observations.

A peroxiredoxin-P38 MAPK scaffold increases MAPK activity by MAP3K-independent mechanisms.

Peroxiredoxins (Prdxs) utilize reversibly oxidized cysteine residues to reduce peroxides and promote H2O2 signal transduction, including H2O2-induced activation of P38 MAPK. Prdxs form H2O2-induced disulfide complexes with many proteins, including multiple kinases involved in P38 MAPK signaling. Here, we show that a genetically encoded fusion between a Prdx and P38 MAPK is sufficient to hyperactivate the kinase in yeast and human cells by a mechanism that does not require the H2O2-sensing cysteine of the Prdx. We demonstrate that a P38-Prdx fusion protein compensates for loss of the yeast scaffold protein Mcs4 and MAP3K activity, driving yeast into mitosis. Based on our findings, we propose that the H2O2-induced formation of Prdx-MAPK disulfide complexes provides an alternative scaffold and signaling platform for MAPKK-MAPK signaling. The demonstration that formation of a complex with a Prdx is sufficient to modify the activity of a kinase has broad implications for peroxide-based signal transduction in eukaryotes.

Atrial Flutter and Left Hemidiaphragmatic Paralysis in the Setting of Lyme Disease.

Lyme disease, caused by a tick-borne spirochete, Borrelia burgdorferi, is the most common vector-borne disease in the United States. Clinical manifestations can include erythema migrans, carditis, facial nerve palsy, or arthritis. A rare complication of Lyme disease is hemidiaphragmatic paralysis. The first case of this complication was documented in 1986, and since then, there have been 16 case reports associating hemidiaphragmatic paralysis with Lyme disease. This is a case of a patient found to be in atrial flutter likely resulting from left hemidiaphragmatic paralysis as a complication of Lyme disease. The patient was a 49-year-old male recently diagnosed with Lyme disease who was treated with a 10-day course of doxycycline and who presented with dyspnea and chest pain. He appeared in acute distress with tachypnea and tachycardia to 169 beats/minute but was not hypoxic. Electrocardiogram (EKG) showed atrial flutter with a rapid ventricular response (RVR). The patient was sent to the emergency department and was treated with intravenous (IV) metoprolol, followed by an IV diltiazem drip, and ultimately converted to normal sinus rhythm. Chest X-ray demonstrated an elevated left hemidiaphragm. Due to concern for Lyme carditis causing tachyarrhythmia, the patient was started on IV ceftriaxone 2 g daily. A transthoracic echocardiogram showed no valvular abnormalities and a normal ejection fraction, thus indicating a low likelihood of carditis. The patient was transitioned to oral doxycycline for an additional 17 days. During the hospital course, a fluoroscopic chest sniff test confirmed the left hemidiaphragmatic paralysis. A chest X-ray completed after two months showed persistent elevation of the left hemidiaphragm and the patient continued to have mild dyspnea. The main lesson from this case is to consider hemidiaphragmatic paralysis as a possible complication of Lyme disease.

pH response of sequence-controlled polyampholyte brushes.

We use molecular simulation to investigate the pH response of sequence-controlled polyampholyte brushes (PABs) with polymer chains consisting of alternating blocks of weakly acidic and basic monomers. Changes in the ionization state, height, lateral structure, and chain conformations of PABs with pH are found to differ qualitatively from those observed for polyelectrolyte brushes. Grafting density has a relatively modest effect on PAB properties. By contrast, monomer sequence strongly affects the pH response, with the extent of the response increasing with the block size. This trend is attributed to strong electrostatic attractions between oppositely charged blocks, which lead to an increase in chain backfolding as block size increases. This behavior is consistent with that observed for polyampholytes with similar monomer sequences in solution in previous studies. Our study shows that monomer sequence can be used to tune the pH response of weak PABs to generate stimuli-responsive surfaces.

In Situ Imaging of Faujasite Surface Growth Reveals Unique Pathways of Zeolite Crystallization.

Zeolite crystallization occurs by complex processes involving a variety of possible mechanisms. The sol gel media used to prepare zeolites leads to heterogeneous mixtures of solution and solid states with diverse solute species. At later stages of zeolite synthesis when growth occurs predominantly from solution, classical two-dimensional nucleation and spreading of layers on crystal surfaces via the addition of soluble species is the dominant pathway. At earlier stages, these processes occur in parallel with nonclassical pathways involving crystallization by particle attachment (CPA). The relative roles of solution- and solid-state species in zeolite crystallization have been a subject of debate. Here, we investigate the growth mechanism of a commercially relevant zeolite, faujasite (FAU). In situ atomic force microscopy (AFM) measurements reveal that supernatant solutions extracted from a conventional FAU synthesis at various times do not result in growth, indicating that FAU growth predominantly occurs from the solid state through a disorder-to-order transition of amorphous precursors. Elemental analysis shows that supernatant solutions are significantly more siliceous than both the original growth mixture and the FAU zeolite product; however, in situ AFM studies using a dilute clear solution with a lower Si/Al ratio revealed three-dimensional growth of surfaces that is distinct from layer-by-layer and CPA pathways. This unique mechanism of growth differs from those observed in studies of other zeolites. Given that relatively few zeolite frameworks have been the subject of mechanistic investigation by in situ techniques, these observations of FAU crystallization raise the question whether its growth pathway is characteristic of other zeolite structures.

Long-Wavelength Fluctuations in Quasi-2D Supercooled Liquids.

We use molecular simulation to characterize the dynamics of supercooled liquids confined in quasi-2D slit geometries. Similar to bulk supercooled liquids, the confined systems exhibit subdiffusive dynamics on intermediate time scales arising from particle localization inside their neighbor cages, followed by an eventual crossover to diffusive behavior as cage rearrangement occurs. The quasi-2D confined liquids also exhibit signatures of long-wavelength fluctuations (LWFs) in the lateral directions parallel to the confining walls, reminiscent of the collective displacements observed in 2D but not 3D systems. The magnitude of the LWFs increases with the lateral dimensions of systems with the same particle volume fraction and confinement length scale, consistent with the logarithmic scaling predicted for 2D Mermin-Wagner fluctuations. The amplitude of the fluctuations is a nonmonotonic function of the confinement length scale because of a competition between caging and strengthening LWFs upon approaching the 2D limit. Our findings suggest that LWFs may play an important role in understanding the behavior of confined supercooled liquids due to their prevalence over a surprisingly broad range of particle densities and confinement length scales.

Anti-Cholestatic Therapy with Obeticholic Acid Improves Short-Term Memory in Bile Duct-Ligated Mice.

Patients with cholestatic liver disease, including those with primary biliary cholangitis, can experience symptoms of impaired cognition or brain fog. This phenomenon remains unexplained and is currently untreatable. Bile duct ligation (BDL) is an established rodent model of cholestasis. In addition to liver changes, BDL animals develop cognitive symptoms early in the disease process (before development of cirrhosis and/or liver failure). The cellular mechanisms underpinning these cognitive symptoms are poorly understood. Herein, the study explored the neurocognitive symptom manifestations, and tested potential therapies, in BDL mice, and used human neuronal cell cultures to explore translatability to humans. BDL animals exhibited short-term memory loss and showed reduced astrocyte coverage of the blood-brain barrier, destabilized hippocampal network activity, and neuronal senescence. Ursodeoxycholic acid (first-line therapy for most human cholestatic diseases) did not reverse symptomatic or mechanistic aspects. In contrast, obeticholic acid (OCA), a farnesoid X receptor agonist and second-line anti-cholestatic agent, normalized memory function, suppressed blood-brain barrier changes, prevented hippocampal network deficits, and reversed neuronal senescence. Co-culture of human neuronal cells with either BDL or human cholestatic patient serum induced cellular senescence and increased mitochondrial respiration, changes that were limited again by OCA. These findings provide new insights into the mechanism of cognitive symptoms in BDL animals, suggesting that OCA therapy or farnesoid X receptor agonism could be used to limit cholestasis-induced neuronal senescence.

Neurosteroid Activation of GABA-A Receptors: A Potential Treatment Target for Symptoms in Primary Biliary Cholangitis?

Background and Aims: A third of patients with primary biliary cholangitis (PBC) experience poorly understood cognitive symptoms, with a significant impact on quality of life (QOL), and no effective medical treatment. Allopregnanolone, a neurosteroid, is a positive allosteric modulator of gamma-aminobutyricacid-A (GABA-A) receptors, associated with disordered mood, cognition, and memory. This study explored associations between allopregnanolone and a disease-specific QOL scoring system (PBC-40) in PBC patients. Method: Serum allopregnanolone levels were measured in 120 phenotyped PBC patients and 40 age and gender-matched healthy controls. PBC subjects completed the PBC-40 at recruitment. Serum allopregnanolone levels were compared across PBC-40 domains for those with none/mild symptoms versus severe symptoms. Results: There were no overall differences in allopregnanolone levels between healthy controls (median = 0.03 ng/ml (IQR = 0.025)) and PBC patients (0.031 (0.42), p = 0.42). Within the PBC cohort, higher allopregnanolone levels were observed in younger patients (r (120) = -0.53, p < 0.001) but not healthy controls (r (39) = -0.21, p = 0.21). Allopregnanolone levels were elevated in the PBC-40 domains, cognition (u = 1034, p = 0.02), emotional (u = 1374, p = 0.004), and itch (u = 795, p = 0.03). Severe cognitive symptoms associated with a younger age: severe (50 (12)) vs. none (60 (13); u = 423 p = 0.001). Conclusion: Elevated serum allopregnanolone is associated with severe cognitive, emotional, and itch symptoms in PBC, in keeping with its known action on GABA-A receptors. Existing novel compounds targeting allopregnanolone could offer new therapies in severely symptomatic PBC, satisfying a significant unmet need.

Nanoparticle dispersion in porous media: Effects of attractive particle-media interactions.

We investigate the effects of physicochemical attractions on the transport of finite-sized particles in three-dimensional ordered nanopost arrays using Stokesian dynamics simulations. We find that weak particle-nanopost attractions negligibly affect diffusion due to the dominance of Brownian fluctuations. Strong attractions, however, significantly hinder particle diffusion due to localization of particles around the nanoposts. Conversely, under flow, attractions significantly enhance longitudinal dispersion at low to moderate Péclet number (Pe). At high Pe, by contrast, advection becomes dominant and attractions weakly enhance dispersion. Moreover, attractions frustrate directional locking at moderate flow rates, and shift the onset of this behavior to higher Pe.

Pharmacological testing of therapeutics using normothermic machine perfusion: A pilot study of 2,4-dinitrophenol delivery to steatotic human livers.

INTRODUCTION: Normothermic machine perfusion (NMP) provides a platform for drug-delivery. However, pharmacological considerations for therapeutics delivered during NMP are scarcely reported. We aimed to demonstrate the ability of NMP as a platform for pharmacological testing, using a drug which increases metabolism (2,4-dinitrophenol; DNP) as an example therapeutic. METHODS: We performed 25 h of NMP on human livers which had been declined for transplant due to steatosis (n = 7). Three livers received a DNP bolus, three were controls, and one received a DNP infusion. RESULTS: Toxicity studies revealed DNP delivery was safe, without hepatotoxic effects. The liver surface temperature was increased in the DNP group (p = 0.046), but no livers suffered hyperthermia-the mechanism of DNP toxicity in vivo. Pharmacokinetic studies revealed DNP elimination with first-order kinetics and 7.7 h half-life (95% CI = 5.1-15.9 hrs). The clearance of DNP in bile was negligible. As expected, DNP significantly increased oxygen consumption (p = 0.023); this increase was closely correlated with perfusate DNP concentration (r2  = 0.975; p = 0.002) and the effect was lost as DNP was eliminated by the liver. A DNP infusion rate, calculated using our pharmacokinetic data, successfully maintained perfusate DNP concentration. DISCUSSION: Detailed pharmacological testing can be performed during NMP. Our therapeutic (DNP) is rapidly eliminated by the ex vivo liver, meaning the drug effect of increased metabolism is only transient. This demonstrates the importance of assessing pharmacokinetics when delivering therapeutics during NMP, especially for prolonged perfusion of organs with established roles in drug elimination. Rigorous pharmacological testing is needed to unlock the potential of NMP as a clinical drug-delivery platform.

The relationship between disease activity and UDCA response criteria in primary biliary cholangitis: A cohort study.

BACKGROUND: Uncertainty exists about how best to identify primary biliary cholangitis (PBC) patients who would benefit from second-line therapy. Existing, purely clinical, ursodeoxycholic acid (UDCA) response criteria accept degrees of liver biochemistry abnormality in responding patients, emerging data, however, suggest that any degree of ongoing abnormality may, in fact, be associated with an increased risk of adverse outcomes. This cohort study explores the link between response status, the biology of high-risk disease and its implications for clinical practice. METHODS: Proteomics, exploring 19 markers previously identified as remaining elevated in PBC following UDCA therapy, were performed on 400 serum samples, from participants previously recruited to the UK-PBC Nested Cohort between 2014 and 2019. All participants had an established diagnosis of PBC and were taking therapeutic doses of UDCA for greater than 12 months. UDCA response status was assessed using Paris 1, Paris 2 and the POISE criteria, with additional analyses using normal liver blood tests stratified by bilirubin level. Statistical analysis using parametric t tests and 1-way ANOVA. FINDINGS: Disease markers were statistically significantly higher in UDCA non-responders than in responders for all the UDCA response criteria, suggesting a meaningful link between biochemical disease status and disease mechanism. For each of the criteria, however, marker levels were also statistically significantly higher in responders with ongoing liver function test abnormality compared to those who had normalised their liver biochemistry. IL-4RA, IL-18-R1, CXCL11, 9 and 10, CD163 and ACE2 were consistently elevated across all responder groups with ongoing LFT abnormality. No statistically significant differences occurred between markers in normal LFT groups stratified by bilirubin level. INTERPRETATION: This study provides evidence that any ongoing elevation in alkaline phosphatase levels in PBC after UDCA therapy is associated with some degree of ongoing disease activity. There was no difference in activity between patients with normal LFT when stratified by bilirubin. These findings suggest that if our goal is to completely control disease activity in PBC, then normalisation of alkaline phosphatase and bilirubin should be the treatment target. This would also simplify messaging around goals of therapy in PBC, benefiting both patients and clinicians. FUNDING: Funding by the UK Medical Research Council (Stratified Medicine Programme) and an independent research grant by Pfizer. The study funders played no role in the study design, data collection, data analyses, data interpretation or manuscript writing.

Spatiotemporal Coke Coupling Enhances para-Xylene Selectivity in Highly Stable MCM-22 Catalysts.

Identifying zeolite catalysts that can simultaneously optimize p-xylene selectivity and feed utilization is critical to toluene alkylation with methanol (TAM). Here, we show that zeolite MCM-22 (MWW) has an exceptional catalyst lifetime in the TAM reaction at high operating pressure, conversion, and selectivity. We systematically probe the catalytic behavior of active sites in distinct topological features of MCM-22, revealing that high p-xylene yield and catalyst stability are predominantly attributed to sinusoidal channels and supercages, respectively. Using a combination of catalyst design and testing, density functional theory, and molecular dynamics simulations, we propose a spatiotemporal coke coupling phenomenon to explain a multistage p-xylene selectivity profile wherein the formation of light coke in supercages initiates the deactivation of unselective external surface sites. Our findings indicate that the specific nature of coke is critical to catalyst performance. Moreover, they provide unprecedented insight into the synchronous roles of distinct topological features giving rise to the exceptional stability and selectivity of MCM-22 in the TAM reaction.

Precrystallization solute assemblies and crystal symmetry.

Solution crystallization is a part of the synthesis of materials ranging from geological and biological minerals to pharmaceuticals, fine chemicals, and advanced electronic components. Attempts to predict the structure, growth rates and properties of emerging crystals have been frustrated, in part, by the poor understanding of the correlations between the oligomeric state of the solute, the growth unit, and the crystal symmetry. To explore how a solute monomer or oligomer is selected as the unit that incorporates into kinks and how crystal symmetry impacts this selection, we combine scanning probe microscopy, optical spectroscopy, and all-atom molecular simulations using as examples two organic materials, olanzapine (OZPN) and etioporphyrin I (EtpI). The dominance of dimeric structures in OZPN crystals has spurred speculation that the dimers preform in the solution, where they capture the majority of the solute, and then assemble into crystals. By contrast, EtpI in crystals aligns in parallel stacks of flat EtpI monomers unrelated by point symmetry. Raman and absorption spectroscopies show that solute monomers are the majority solute species in solutions of both compounds. Surprisingly, the kinetics of incorporation of OZPN into kinks is bimolecular, indicating that the growth unit is a solute dimer, a minority solution component. The disconnection between the dominant solute species, the growth unit, and the crystal symmetry is even stronger with EtpI, for which the (010) face grows by incorporating monomers, whereas the growth unit of the (001) face is a dimer. Collectively, the crystallization kinetics results with OZPN and EtpI establish that the structures of the dominant solute species and of the incorporating solute complex do not correlate with the symmetry of the crystal lattice. In a broader context, these findings illuminate the immense complexity of crystallization scenarios that need to be explored on the road to the understanding and control of crystallization.

Signatures of sluggish dynamics and local structural ordering during ice nucleation.

We investigate the microscopic pathway of spontaneous crystallization in the ST2 model of water under deeply supercooled conditions via unbiased classical molecular dynamics simulations. After quenching below the liquid-liquid critical point, the ST2 model spontaneously separates into low-density liquid (LDL) and high-density liquid phases, respectively. The LDL phase, which is characterized by lower molecular mobility and enhanced structural order, fosters the formation of a sub-critical ice nucleus that, after a stabilization time, develops into the critical nucleus and grows. Polymorphic selection coincides with the development of the sub-critical nucleus and favors the formation of cubic (Ic) over hexagonal (Ih) ice. We rationalize polymorphic selection in terms of geometric arguments based on differences in the symmetry of second neighbor shells of ice Ic and Ih, which are posited to favor formation of the former. The rapidly growing critical nucleus absorbs both Ic and Ih crystallites dispersed in the liquid phase, a crystal with stacking faults. Our results are consistent with, and expand upon, recent observations of non-classical nucleation pathways in several systems.

Macrophage scavenger receptor 1 mediates lipid-induced inflammation in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Obesity-associated inflammation is a key player in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the role of macrophage scavenger receptor 1 (MSR1, CD204) remains incompletely understood. METHODS: A total of 170 NAFLD liver biopsies were processed for transcriptomic analysis and correlated with clinicopathological features. Msr1-/- and wild-type mice were subjected to a 16-week high-fat and high-cholesterol diet. Mice and ex vivo human liver slices were treated with a monoclonal antibody against MSR1. Genetic susceptibility was assessed using genome-wide association study data from 1,483 patients with NAFLD and 430,101 participants of the UK Biobank. RESULTS: MSR1 expression was associated with the occurrence of hepatic lipid-laden foamy macrophages and correlated with the degree of steatosis and steatohepatitis in patients with NAFLD. Mice lacking Msr1 were protected against diet-induced metabolic disorder, showing fewer hepatic foamy macrophages, less hepatic inflammation, improved dyslipidaemia and glucose tolerance, and altered hepatic lipid metabolism. Upon induction by saturated fatty acids, MSR1 induced a pro-inflammatory response via the JNK signalling pathway. In vitro blockade of the receptor prevented the accumulation of lipids in primary macrophages which inhibited the switch towards a pro-inflammatory phenotype and the release of cytokines such as TNF-ɑ. Targeting MSR1 using monoclonal antibody therapy in an obesity-associated NAFLD mouse model and human liver slices resulted in the prevention of foamy macrophage formation and inflammation. Moreover, we identified that rs41505344, a polymorphism in the upstream transcriptional region of MSR1, was associated with altered serum triglycerides and aspartate aminotransferase levels in a cohort of over 400,000 patients. CONCLUSIONS: Taken together, our data suggest that MSR1 plays a critical role in lipid-induced inflammation and could thus be a potential therapeutic target for the treatment of NAFLD. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) is a chronic disease primarily caused by excessive consumption of fat and sugar combined with a lack of exercise or a sedentary lifestyle. Herein, we show that the macrophage scavenger receptor MSR1, an innate immune receptor, mediates lipid uptake and accumulation in Kupffer cells, resulting in liver inflammation and thereby promoting the progression of NAFLD in humans and mice.

Secretory immunoglobulin A in preterm infants: determination of normal values in breast milk and stool.

BACKGROUND: IgA and its secretory form sIgA impact protection from infection and necrotising enterocolitis but little is known about quantities in preterm mums own milk (MOM) or infant stool, onset of endogenous production in the preterm gut, and what affects these. METHODS: We measured by ELISA in MOM and stool from healthy preterm infants total IgA and sIgA longitudinally and additionally in MOM fresh, refrigerated, frozen, and after traversing feeding systems. RESULTS: In 42 MOM (median gestation 26 weeks), we showed total IgA levels and sIgA were highest in colostrum, fell over 3 weeks, and were not impacted by gestation. Median IgA values matched previous term studies (700 mcg/ml). In MOM recipients stool IgA was detected in the first week, at around 30% of MOM quantities. Formula fed infants did not have detectable stool IgA until the third week. Levels of IgA and sIgA were approximately halved by handling processes. CONCLUSIONS: MOM in the 3 weeks after preterm delivery contains the highest concentrations of IgA and sIgA. Endogenous production after preterm birth occurs from the 3 week meaning preterm infants are dependent on MOM for IgA which should be optimised. Routine NICU practices halve the amount available to the infant. IMPACT: (Secretory) Immunoglobulin A (IgA) is present in colostrum of maternal milk from infants as preterm as 23-24 weeks gestational age, falling over the first 3 weeks to steady levels similar to term. Gestation at birth does not impact (secretory) IgA levels in breast milk. IgA is present in very preterm infant stools from maternal milk fed infants from the first week of life, but not in formula milk fed preterm infants until week three, suggesting endogenous production from this point. Refrigeration, freezing, and feeding via plastic tubing approximately halved the amount of IgA available.