SEM and LM insights: Spore morphology and its taxonomic significance in Sino Himalayan, Malesian, and European elements of Pteridaceae.

The Pteridaceae family, known for its taxonomic complexity, presents challenges in identification due to high variability among its species. This study investigates the spore morphology employing both SEM and LM techniques in 10 Pteridaceae taxa phytogeographicaly Sino-Himalayan, Malesian, and European elements in Pakistan. The taxa include Adiantum capillus-veneris, A. incisum, A. venustum, Aleuritopteris bicolor, Oeosporangium nitidulum, O. pteridioides, Onychium cryptogrammoides, O. vermae, Pteris cretica, and P. vittata. The objective is to assess their taxonomic relevance and develop a spore-based taxonomic key. Findings indicate differences in spore shape, sizes, exospore thickness, and in surface ornamentation highlighting the potential for taxonomic differentiation. Spores are trilete, and notable differences are observed in the dimension of spores in both distal and proximal sides. Equatorial dimensions vary between 35 and 50 µm, while the polar diameter ranges from 29 to 50 µm. SEM revealed different spore ornamentation types that show several useful characteristics establishing valuable taxonomic variations. The studied Adiantum taxa feature a perispore with tubercules and a micro-granulose surface. The spores of examined Oeosporangium and Aleuritopteris taxa shows cristate sculptures with variable ornamentations. Both species of Onychium have tuberculate-pleated tubercles with sinuous folds on both distal and proximal sides. The surface ornamentation among examined Pteris taxa show variability. PCA analysis indicated that spore quantitative data identified distinct groups, underscoring taxonomic significance. Nevertheless, there was variation observed in surface ornamentation and spore shape, indicating the potential for discrimination among taxa. RESEARCH HIGHLIGHTS: Spore morphology of 10 Pteridaceae taxa has been investigated through LM and SEM. Investigated species shows differences in spore shape, sizes, exospore thickness, and in surface ornamentation. Ornamentation on the perispore provides several valuable characteristics, establishing useful taxonomic distinctions. Spore morphological analysis is effective at the generic level, with minor distinctions discernible at the species level.

Staff radiation doses during Sentinel Lymph Node procedure of breast cancer from injection to surgeon.

The Sentinel Lymph Node (SLN) or Sentinel Lymph Node Biopsy (SLNB) technique involves various professionals from different departments in clinical settings to manage breast cancer patients properly. Tracing the nodular involvement of breast cancer patients requires radiation source Tc99m labeled with colloidal albumin to be injected at the tumor site. The patient becomes a radiation source for a sufficient time, which concerns the Nuclear Medicine (NM) and surgical staff. The study aims to provide the radiation doses of staff in the NM department during the SLN scintigraphy procedure and obtain an empirical model for calculating the radiation doses to staff in the surgical department from that particular patient. Radiation doses in SLN technique for breast cancer patients are minimal, and a sufficient number of SLN biopsy procedures can be performed by hospital staff within the category of non-radiation workers.

Care Coordination Between Family Physicians and Palliative Care Physicians for Patients With Cancer: Results of a Quality Improvement Initiative.

PURPOSE: At our institution's cancer palliative care (PC) clinic, new referrals from oncologists were scheduled for consultation and ongoing follow-up by PC physicians without input from the patients' family physicians (FPs). FPs reported that they felt out of the loop. We implemented a quality improvement (QI) initiative aimed at systematically facilitating care coordination between FPs and PC physicians. METHODS: A coordination toolkit was sent from the PC physician to the FP whenever the PC physician received a consultation request from an oncologist. The toolkit included an introduction to the PC physician team; an opportunity for the FP to choose how best to collaborate with PC physicians to meet the patient's PC needs; and contact information for access to 24/7 PC physician support. Responses from FPs regarding their preferred level of engagement with PC determined further care planning in the clinic. We measured feasibility, response rate, and qualitative surveys of FPs about the usefulness of the intervention. RESULTS: Two hundred fourteen new consultations were eligible for a standardized letter over the 6-month implementation period. Feasibility for sending the toolkit was 90.0% and response rate for collaborative care preference from FPs was 86.0%, with median response time of 3-4 days. 78.9% of FPs indicated they would prefer ongoing consultative care by the PC physician, while 18.6% indicated that PC physician consultation was not needed, or that the FP would provide primary PC after a one-time PC physician consultation. CONCLUSION: We successfully implemented a QI initiative to improve care coordination between FPs and PC physicians for patients with cancer. The coordination toolkit can protect the patient-FP primary PC relationship and optimize specialist PC resource utilization for complex patients.

Mitigation effect of alpha-tocopherol and thermo-priming in Brassica napus L. under induced mercuric chloride stress.

Soil pollution with heavy metals has grown to be a big hassle, leading to the loss in farming production particularly in developing countries like Pakistan, where no proper channel is present for irrigation and extraction of these toxic heavy metals. The present study aims to ameliorate the damages caused by heavy metal ions (Hg-Mercury) on rapeseed (Brassica napus L.) via a growth regulator (α-tocopherol 150 mg/L) and thermopriming technique at 4 °C and 50 °C to maintain plant agronomical and physiological characteristics. In pot experiments, we designed total of 11 treatments viz.( T0 (control), T1 (Hg4ppm), T2 (Hg8ppm), T3 (Hg4ppm + 4 °C), T4 (Hg4ppm + 4 °C + tocopherol (150 m/L)), T5 (Hg4ppm + 50 °C), T6 (Hg4ppm + 50 °C + tocopherol (150 mg/L)), T7 (Hg8ppm + 4 °C), T8 (Hg8ppm + 4 °C + tocopherol (150 mg/L)), T9 (Hg8ppm + 50 °C), T10 (Hg8ppm + 50 °C + tocopherol (150 mg/L) the results revealed that chlorophyll content at p < 0.05 with growth regulator and antioxidant enzymes such as catalase, peroxidase, and malondialdehyde enhanced up to the maximum level at T5 = Hg4ppm + 50 °C (50 °C thermopriming under 4 ppm mercuric chloride stress), suggesting that high temperature initiate the antioxidant system to reduce photosystem damage. However, protein, proline, superoxide dismutase at p < 0.05, and carotenoid, soluble sugar, and ascorbate peroxidase were increased non-significantly (p > 0.05) 50 °C thermopriming under 8 ppm high mercuric chloride stress (T9 = Hg8ppm + 50 °C) representing the tolerance of selected specie by synthesizing osmolytes to resist oxidation mechanism. Furthermore, reduction in % MC (moisture content) is easily improved with foliar application of α-tocopherol and 50 °C thermopriming and 4 ppm heavy metal stress at T6 = Hg4ppm + 50 °C + α-tocopherol (150 mg/L), with a remarkable increase in plant vigor and germination energy. It has resulted that the inhibitory effect of only lower concentration (4 ppm) of heavy metal stress was ameliorated by exogenous application of α-tocopherol and thermopriming technique by synthesizing high levels of proline and antioxidant activities in maintaining seedling growth and development on heavy metal contaminated soil.

Study Comparing Topical Ivermectin Versus Topical Permethrin in the Treatment of Scabies.

Introduction Scabies is a highly contagious skin disease caused by an ectoparasite mite called Sarcoptes scabiei. Ivermectin and permethrin have been commonly used for the treatment of scabies. However, topical ivermectin has been compared to other treatment modalities to a lesser extent. Objective This study aimed to compare the efficacy of topical ivermectin versus topical permethrin in the treatment of uncomplicated scabies. Methods 354 patients with scabies attending the dermatology outpatient department of Pak Emirates Military Hospital Rawalpindi were enrolled. Patients were divided into two groups randomly. The first group and their family contacts received 1% ivermectin lotion whereas the other received 5% permethrin lotion. Patients were evaluated at the end of the second and the fourth week. Results At the end of the second week, initial follow-up showed that 97 out of 159 patients (61.0%) in the ivermectin 1% group, and 107 out of 159 patients (67.3%) in the permethrin 5% group had achieved clinical cure (P=0.24). On the final follow-up at the end of Week 4, the cure rate amounted to 85.5% (136 of 159 patients) in the ivermectin group and 89.9% (143 of 159 patients) in the permethrin group. Differences among both groups remained statistically insignificant (P=0.23). Conclusions The use of ivermectin 1% versus permethrin 5% as topical therapy showed almost identical results for the treatment of uncomplicated scabies. Side effects were minimal and there were no significant differences observed in patients with regard to compliance among both the groups.

Potential drug-drug interactions in patients presenting with osteoarthritis to community orthopaedic clinics of Abbottabad, Khyber Pakhtunkhwa: A cross-sectional study.

OBJECTIVES: Osteoarthritis (OA) causes long-term pain and disorders of lower extremities. Paracetamol is the drug of choice; however, NSAIDs, opioids and steroids are frequently employed in the symptomatic relief of OA. The prescribing of multiple analgesics leads to potential drug-drug interactions (pDDIs). The primary objective of this study was to identify the prevalence and predictors of pDDIs in OA. MATERIALS AND METHODS: A total of three hundred and eighty-six patients, either newly diagnosed or with a history of OA, were enrolled for this cross-sectional study. Data regarding patients' demographics, clinical characteristics and prescribed medications were recorded from the prescriptions and examined for the pDDIs using Medscape multidrug interaction checker. RESULTS: Among 386, most patients were females (53.4%). The most prevalent diagnoses were knee OA (39.7%) and unspecified OA (31.3%). Paracetamol and topical NSAIDs were underprescribed whereas an oral NSAID, Diclofenac, was the most frequently used drug in OA. Total of 109 pDDIs were found in 386 prescriptions, of which 63.3% DDIs were categorised as moderate, followed by minor (34.9%) and major (1.8%). CONCLUSIONS: This study finds a prevalence of DDIs and polypharmacy among the OA patients. Collaborative efforts among healthcare providers, pharmacists, and patients themselves are essential to optimize medication regimens and minimize the polypharmacy including the associated risks as well as DDIs.

Application of spore morphology to solve identification problems in certain species of family Dryopteridaceae from Malakand Division, Pakistan.

Here we investigate the morphology of the spores of 12 native taxa of Dryopteridaceae that grow in Malakand division, Pakistan; most of these species not fully described before. This study intends to survey the taxonomic significance of spore morphological features and their variation useful for species identification in 7 species of Polystichum and 5 taxa of Dryopteris. The spores examinations were accomplished utilizing a light microscope (LM), and scanning electron microscope (SEM). The spores are monolete, medium-sized, with variable shapes in both equatorial and polar views. The mean estimation of equatorial diameter ranging from 28.3 to 58.3 µm and the polar diameter varied from 27.6 to 45.8 µm. The exospore thickness ranges between 1.6 to 3.8 µm, and the thickness of perispore is 0.8-6.7 µm. The perispore is perforated, ornamentation elements occur in different ways in the studied taxa of Polystichum. In investigated Dryopteris taxa, rugate perispore type is found. Results of multivariate statistical analysis (UPGMA, and PCA) established on quantitative and qualitative morphological traits of spores demonstrate that the species form distinct groups, not reflecting any phylogenetic relationships. A key to the spores types is provided for species identification, which dependent on the diagnostic characters of spores. The results of this study indicate that variation of spore morphological traits; in particular, the ornamentation and perispore characteristics, which is surface texture, have diagnostic value at the species level. Our data will help in the identification of spores of the family Dryopteridaceae in palynological and taxonomic studies.

Spore morphology and leaf epidermal anatomy as a taxonomic source in the identification of Asplenium species from Malakand division Pakistan.

Several studies have demonstrated the usefulness of leaf epidermal, and spore morphological characters in the taxonomy of genus Asplenium. However, works on the Pakistani species of Asplenium are not existent. With the objective to verify the efficacy of leaf epidermal and spore morphological traits, the leaf epidermis and spore morphology in nine Asplenium species occurring in Malakand division was studied. The spores were studied under light microscope (LM) and scanning electron microscope (SEM), whereas for leaf epidermal anatomy, the LM was used. The spores are monolete, ellipsoidal in shape, the equatorial and polar diameter ranges between 28.3 and 50.2 × 27.6 and 45.8 µm. The exospore thickness varied from 0.5 to 3.8 µm. The perispore is 0.8-3.5 µm thick, ornamented, and morphologically variable. The spores characters specifically the perispore ornamentation are useful in distinguishing species within the genus. The irregular spore shape and unusual development of perispore wall surface characterize aborted spores in the species of Asplenium × alternifolium. The most informative quantitative characters appeared to be length and width of epidermal cells, and length and width of stomata were useful to distinguish species. The most significant qualitative characters to distinguish species were the anticlinal wall pattern. Our study has shown that considerable variations exist in the leaf epidermis of Asplenium species, at least some of which has taxonomic significance. We confirmed the prevalent taxonomic value of stomatal and epidermal cells traits. The leaf epidermal anatomy and spore morphological features showed to be a good source of information for taxonomy of the genus Asplenium.

Seed micromorphology and its taxonomic evidence in subfamily Alsinoideae (Caryophyllaceae).

Seed micromorphology of 13 species, belonging to four genera of subfamily Alsinoideae (Caryophyllaceae) were investigated with scanning electron microscopy (SEM), in order to assess their diagnostic significance at generic level and provide additional evidence on species delimitation, as well as correct identification and phylogenetic position. Genera and species of subfamily Alsinoideae exhibit great variation in ultrastructure and a high diversity of novel micromorphological characters were observed. Variation in seed shape, color, hilum, anticlinal wall, epidermal cell, cell surface, margins, and quantitative characters as length and width were studied in detail, compared, illustrated, and their taxonomic significant were discussed. Seed shapes of the species were classified as reniform, round, angular, subcircular, subreniform, and elliptical pyriform, with sub-central, central, basal, and nearly basal hilum. Wavy, irregular, tetragonal, and elongated epidermal cells structure has been observed as an exomorphological character. The present findings show that the micromorphology of subfamily Alsinoideae provides taxonomic information and is helpful to distinguish different species. The results also explained that SEM morphology of seeds provide important data about affinity among taxa and give potential characters in delimitation of members of subfamily Alsinoideae at generic and species level. A principal component analysis allowed to highlight the most outsiders among seed micromorphology with a possible explanation. Taxonomic keys were developed based on micromorphological characters to delimit the species and useful for their quick identification within subfamily Alsinoideae.

Leaf epidermal micromorphology and its implications in systematics of certain taxa of the fern family Pteridaceae from Northern Pakistan.

The present study is insights into foliar epidermal anatomy for characterizing clades, and their utility in taxonomic segregation of certain species of Pteridaceae from Northern Pakistan. The leaf epidermal anatomy of 10 species of Pteridaceae representing four genera were examined using light and scanning electron microscope. A micromorphological matrix was constructed for eight qualitative and 12 quantitative characters. unweighted pair group method with arithmetic means and principal components analysis statistical analysis were performed to test the validity of foliar epidermal anatomical features as method of separating species and genera, and phylogenetic clusters among species are constructed using qualitative and quantitative traits. The qualitative characters described here are shape of epidermal cells, stomata, guard cell and subsidiary cells, anticlinal wall pattern, and trichomes types which is helpful in defining groups within Pteridaceae. In addition, the size of stomata, guard cells, subsidiary cells, stomatal pore epidermal cells, and trichomes are quantitatively analyzed. All species have hypostomatic leaves. Two types of stomata were observed in studied species, anomocytic and polocytic. Anomocytic stomata were observed in three genera namely: Adiantum, Onychium, and Chielanthes whereas Pteris can be discriminated from other genera by its polocytic stomata. On the basis of multivariate analysis present study does provides sufficient information on the taxonomic importance of foliar anatomy which validate its efficacy in species and genera discrimination. From result obtained here it is further possible to use leaf micromorphologic data in ferns phylogeny and providing basis for future taxonomic delimitation in other taxa.

Foliar epidermal micromorphology and its taxonomic implications in some selected species of Athyriaceae.

For the robust identification of taxonomically complex fern family like Athyriaceae, light and scanning electron microscopy is significance implications. This article present first microscopic investigation of foliar micromorphology of 3 genera and 10 species belonging to Athyriaceae namely, Athyrium, Deparia, and Diplazium were collected from different localities in Malakand Division, Northern Pakistan. In present study we compare foliar micromorphology of all 10 species using standard protocols of light microcopy (LM) and scanning electron microscopy. Qualitative micromorphological variations in shape of epidermal cells, anticlinal wall pattern, stomatal type and shape, stomatal pore shape, guard cells shape, and trichomes types were studied. In addition, some quantitative characters were also studied and data were statistically analyzed in epidermal cell size, stomatal size, stomatal pore size, stomatal density, and stomatal index. The pivotal result of study include; shape of epidermal cell in all species is irregular on both abaxial and adaxial surfaces. The anticlinal walls are sinuous in most of the species but some species have irregular lobed and broadly lobed wall. Leaves are hypostomatic in all studied species. Two main categories of stomatal type were found: polocytic and anomocytic. Unicellular nonglandular trichomes were observed in only one species Athyrium mackinnoni. The variation in foliar micromorphological characters between the genera and within the species was useful in identification and classification and have potential taxonomic significance for species differentiation. An identification key using micromorphological characters are provided to distinguish genera and species.

Pollen morphology of subfamily Caryophylloideae (Caryophyllaceae) and its taxonomic significance.

The Pollen morphology of subfamily Caryophylloideae (Caryophyllaceae) from different phytogeographical region of Pakistan has been evaluated. In this research, 16 species belong to 6 genera of subfamily Caryophylloideae have been studied using light (LM) and scanning electron microscopy (SEM) for both qualitative and quantitative characters. Different palyno-morphological features were observed including; pollen ornamentation, pore ornamentation, echini arrangement, echinidensity, number of pori, size of polar and equatorial diameter, P/E ratio, exine thickness, and size of pore were studied. The palyno-morphological characters of subfamily Caryophylloideae have taxonomically significant in identification and delimitation of species. Two pollen types, i.e., subspheroidal (15 species) and prolate (one species Vaccaria hispanica) were observed. Microechinate-punctate and microechinate-perforate pollen ornamentation were examined. Two pollen types of pori, i.e., prominent (5 species) and sunken (11 species) ornamentation have been reported. Three types of echini arrangement have been reported irregular, regular and rather regular, while three type of echinidensity (i.e., medium, dense and sparse) were observed. Pori numbers were found different in different species range from 5 in Silene apetala to 19-35 in Silene vulgaris. Based on different palyno-morphological characters, taxonomic key was develop for quick and accurate identification. The quantitative data were processed using SPSS software for average, minimum, maximum, and standard error.

Aminolevulinic acid and nitric oxide regulate oxidative defense and secondary metabolisms in canola (Brassica napus L.) under drought stress.

To minimize the damaging effects of stresses, plant growth regulators (PGRs) are widely used to sustain the plant life under stress-prone environments. So, a study was carried out to evaluate the response of two canola (Brassica napus L.) cultivars, Dunkeld and Cyclone, to foliar-applied two potential PGRs, nitric oxide (NO) and 5-aminolevulinic acid, under water deficit conditions. In this study, the levels of NO and ALA used were 0.02 and 0.895 mM, respectively. Plants of both canola cultivars were subjected to control (100% field capacity) and water deficit (60% field capacity). Drought stress significantly decreased growth, chlorophyll pigments, relative water contents (RWC), and soluble proteins, while it increased relative membrane permeability (RMP), proline, glycinebetaine (GB), malondialdehyde (MDA), total phenolics, and activities of catalase (CAT) and peroxidase (POD) enzymes in both cultivars. Foliar application of PGRs improved growth, chlorophyll a, GB, total phenolics, CAT activity, and total soluble proteins, while it decreased RMP, MDA, and POD activity in both canola cultivars. Other physio-biochemical attributes such as chlorophyll b, RWC, hydrogen peroxide (H2O2) and proline contents as well as superoxide dismutase (SOD) activity remained unaffected due to application of PGRs. So, the results of the present study suggest that exogenous application of NO and ALA could be useful to enhance the drought tolerance of canola plants by up-regulating the oxidative defense system, osmoprotectant accumulation, and minimizing the lipid peroxidation.

Acute Effects of Viral Exposure on P-Glycoprotein Function in the Mouse Fetal Blood-Brain Barrier.

BACKGROUND/AIMS: Viral infection during pregnancy is known to affect the fetal brain. The toll-like receptor (TLR)-3 is a pattern recognition receptor activated by viruses known to elicit adverse fetal neurological outcomes. The P-glycoprotein (P-gp) efflux transporter protects the developing fetus by limiting the transfer of substrates across both the placenta and the fetal blood-brain barrier (BBB). As such, inhibition of P-gp at these blood-barrier sites may result in increased exposure of the developing fetus to environmental toxins and xenobiotics present in the maternal circulation. We hypothesized that viral exposure during pregnancy would impair P-gp function in the placenta and in the developing BBB. Here we investigated whether the TLR-3 ligand, polyinosinic:polycytidylic acid (PolyI:C), increased accumulation of one P-gp substrate in the fetus and in the developing fetal brain. METHODS: Pregnant C57BL/6 mice (GD15.5) were injected (i.p.) with PolyI:C (5 mg/kg or 10 mg/kg) or vehicle (saline). [3H]digoxin (P-gp substrate) was injected (i.v.) 3 or 23h post-treatment and animals were euthanized 1h later. Maternal plasma, 'fetal-units' (fetal membranes, amniotic fluid and whole fetus), and fetal brains were collected. RESULTS: PolyI:C exposure (4h) significantly elevated maternal plasma IL-6 (P<0.001) and increased [3H]digoxin accumulation in the fetal brain (P<0.05). In contrast, 24h after PolyI:C exposure, no effect on IL-6 or fetal brain accumulation of P-gp substrate was observed. CONCLUSION: Viral infection modeled by PolyI:C causes acute increases in fetal brain accumulation of P-gp substrates and by doing so, may increase fetal brain exposure to xenobiotics and environmental toxins present in the maternal circulation.

Astrocyte-mediated regulation of multidrug resistance p-glycoprotein in fetal and neonatal brain endothelial cells: age-dependent effects.

Brain endothelial cells (BECs) form a major component of the blood-brain barrier (BBB). In late gestation, these cells express high levels of the multidrug transporter p-glycoprotein (P-gp; encoded by Abcb1), which prevents the passage of an array of endogenous factors and xenobiotics into the fetal brain. P-gp levels in the BECs increase dramatically in late gestation, coincident with astrocyte differentiation. However, the role of astrocytes in modulating P-gp in the developing BBB is unknown. We hypothesized that factors produced by astrocytes positively regulate P-gp in BECs. Astrocytes and BECs were isolated from fetal and postnatal guinea pigs. Levels of Abcb1 mRNA and P-gp were increased in BECs co-cultured with astrocytes compared to BECs in monoculture. Moreover, postnatal astrocytes enhanced P-gp function in fetal BECs but fetal astrocytes had no effect on postnatal BECs. These effects were dependent on secreted proteins with a molecular weight in the range of 3-100 kDa. LC/MS-MS revealed significant differences in proteins secreted by fetal and postnatal astrocytes. We propose that astrocytes are critical modulators of P-gp at the developing BBB. As such, aberrations in astrocyte maturation, observed in neurodevelopmental disorders, will likely decrease P-gp at the BBB. This would allow increased transfer of P-gp endogenous and exogenous substrates into the brain, many of which have neurodevelopmental consequences.

Glucocorticoids modify effects of TGF-ß1 on multidrug resistance in the fetal blood-brain barrier.

Transforming growth factor-ß1 (TGF-ß1) increases P-glycoprotein (P-gp; encoded by Abcb1) activity in fetal brain endothelial cells (BECs). P-gp is important for fetal brain protection against xenobiotics including synthetic glucocorticoids (sGC). We hypothesized that antenatal sGC would modify P-gp responsiveness to TGF-ß1 in fetal BECs. Pregnant guinea pigs were treated with dexamethasone or vehicle (N = 5/group) on gestational day (GD) 48-49 and BECs derived on GD50. In BECs from control fetuses, TGF-ß1 increased Abcb1 mRNA and P-gp function, by approximately 5-fold and 55% respectively, as well as tight junction function. In contrast, TGF-ß1 had no effect on these parameters in BECs from sGC-exposed fetuses. Moreover, levels of TGF-ß1 responsive gene, Smad7, were increased 3-fold in BECs from control fetuses after TGF-ß1 but not in sGC-exposed fetuses. In conclusion, antenatal sGC alters responsiveness to TGF-ß1 in fetal BECs. This study has identified novel mechanisms by which TGF-ß1 and sGC modulate fetal brain protection against xenobiotics and other P-gp substrates.

The ontogeny of P-glycoprotein in the developing human blood-brain barrier: implication for opioid toxicity in neonates.

BACKGROUND: Neonates have been shown to have a heightened sensitivity to the central depressive effects of opioids compared to older infants and adults. The limited development of P-glycoprotein (P-gp) may limit the ability of the neonate to efflux morphine from the brain back to the systemic circulation. The objective of the study was to determine the ontogeny of P-gp in the human brain. METHODS: Postmortem cortex samples from gestational age (GA) 20-26 wk, GA 36-40 wk, postnatal age (PNA) 0-3 mo, PNA 3-6 mo, and adults were immunostained for P-gp. RESULTS: The intensity of P-gp staining in adults was significantly higher compared to at GA 20-26 wk (P < 0.05), GA 36-40 wk (P < 0.05), and PNA 0-3 mo (P < 0.05). P-gp intensity at GA 20-26 wk (P < 0.05), GA 36-40 wk (P < 0.05), and PNA 0-3 mo (P < 0.05) was significantly lower compared to at PNA 3-6 mo. CONCLUSION: P-gp expression in the brain is limited at birth, increases with postnatal maturation, and reaches adult levels at ~3-6 mo of age. Given the immaturity of blood-brain barrier (BBB) P-gp after birth, morphine may concentrate in the brain. This provides mechanistic support to life threatening opioid toxicity seen with maternal codeine use during breastfeeding.

The multidrug resistance 1 gene Abcb1 in brain and placenta: comparative analysis in human and guinea pig.

The Multidrug Resistance 1 (MDR1; alternatively ABCB1) gene product P-glycoprotein (P-gp), an ATP binding cassette transporter, extrudes multiple endogenous and exogenous substrates from the cell, playing an important role in normal physiology and xenobiotic distribution and bioavailability. To date, the predominant animal models used to investigate the role of P-gp have been the mouse and rat, which have two distinct genes, Abcb1a and Abcb1b. In contrast, the human has a single gene, ABCB1, for which only a single isoform has been validated. We and others have previously shown important differences between Abcb1a and Abcb1b, limiting the extrapolation from rodent findings to the human. Since the guinea pig has a relatively long gestation, hemomonochorial placentation and neuroanatomically mature offspring, it is more similar to the human, and may provide a more comparable model for investigating the regulation of P-gp in the brain and placenta, however, to date, the Abcb1 gene in the guinea pig remains to be characterized. The placenta and fetal brain are barrier sites that express P-gp and that play a critical role of protection of the fetus and the fetal brain from maternally administered drugs and other xenobiotics. Using RNA sequencing (RNA-seq), reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (QPCR) to sequence the expressed isoforms of guinea pig Abcb1, we demonstrate that like the human, the guinea pig genome contains one gene for Abcb1 but that it is expressed as at least three different isoforms via alternative splicing and alternate exon usage. Further, we demonstrate that these isoforms are more closely related to human than to rat or mouse isoforms. This striking, overall similarity and evolutionary relatedness between guinea pig Abcb1 and human ABCB1 indicate that the guinea pig represents a relevant animal model for investigating the function and regulation of P-gp in the placenta and brain.

TGF-ß1 regulation of multidrug resistance P-glycoprotein in the developing male blood-brain barrier.

P-glycoprotein (P-gp), an efflux transporter encoded by the abcb1 gene, protects the developing fetal brain. Levels of P-gp in endothelial cells of the blood-brain barrier (BBB) increase dramatically during the period of peak brain growth. This is coincident with increased release of TGF-ß1 by astrocytes and neurons. Although TGF-ß1 has been shown to modulate P-gp activity in a number of cell types, little is known about how TGF-ß1 regulates brain protection. In the present study, we hypothesized that TGF-ß1 increases abcb1 expression and P-gp activity in fetal and postnatal BBB in an age-dependent manner. We found TGF-ß1 to potently regulate abcb1 mRNA and P-gp function. TGF-ß1 increased P-gp function in brain endothelial cells (BECs) derived from fetal and postnatal male guinea pigs. These effects were more pronounced earlier in gestation when compared with BECs derived postnatally. To investigate the signaling pathways involved, BECs derived at gestational day 50 and postnatal day 14 were exposed to ALK1 and ALK5 inhibitors and agonists. Through inhibition of ALK5, we demonstrated that ALK5 is required for the TGF-ß1 effects on P-gp function. Activation of ALK1, by the agonist BMP-9, produced similar results to TGF-ß1 on P-gp function. However, TGF-ß1 signaling through the ALK1 pathway is age-dependent as dorsomorphin, an ALK1 inhibitor, attenuated TGF-ß1-mediated effects in BECs derived at postnatal day 14 but not in those derived at gestational day 50. In conclusion, TGF-ß1 regulates P-gp at the fetal and neonatal BBB and both ALK5 and ALK1 pathways are implicated in the regulation of P-gp function. Aberrations in TGF-ß1 levels at the developing BBB may lead to substantial changes in fetal brain exposure to P-gp substrates, triggering consequences for brain development.