Leishmania infantum: stage-specific activity of pentavalent antimony related with the assay conditions

The determination of the intrinsic sensitivity of Leishmania strains to pentavalent antimonials in clinical trials, before treatment is begun, is essential in order to avoid failures and to allow alternative drugs to be chosen. A comparative study of SbV activity on promastigotes, axenic amastigote-like cells, and intracellular amastigotes of Leishmania infantum, when administered in the form of meglumine antimoniate and free, in hydrochloric solution, was performed. Results indicate that the conditions under which the promastigotes were cultured affect the IC(50) obtained, although results were homogeneous when the products were assayed on axenic-like and intracellular amastigotes. The IC(50) obtained for SbV in the form of meglumine antimoniate or in hydrochloric solution on promastigotes cultured in Schneider's medium depends on the growth rate of the culture and therefore could be regulated by modifying the fetal calf serum concentration in the medium. The pH of the culture medium strongly affected the activity of meglumine antimoniate but not that of the SbV hydrochloric solution on promastigotes cultured in Schneider's medium. This influence of pH was observed to a much lesser extent when promastigotes were cultured on M199 or RPMI media. In homogeneous culture conditions, which included the regulation of the promastigote growth rate through the heat-inactivated fetal calf serum concentration in the medium and the dilution of the meglumine antimoniate with Schneider's medium at pH 6.5, the activity of SbV, free or in the form of meglumine antimoniate, was the same in promastigotes, intracellular amastigotes, and axenic amastigote-like cells.     

A behavioural study of neuroglobin-overexpressing mice under normoxic and hypoxic conditions

Neuroglobin (Ngb), a neuron-specific heme-binding protein that binds O₂, CO and NO reversibly, and promotes in vivo and in vitro cell survival after hypoxic and ischaemic insult. Although the mechanisms of this neuroprotection remain unknown, Ngb might play an important role in counteracting the adverse effects of ischaemic stroke and cerebral hypoxia. Several Ngb overexpressing mouse models have confirmed this hypothesis; however, these models were not yet exposed to in-depth behavioural characterisations. To investigate the potential changes in behaviour due to Ngb overexpression, heterozygous mice and wild type (WT) littermates were subjected to a series of cognitive and behavioural tests (i.e., the SHIRPA primary screening, the hidden-platform Morris water maze, passive avoidance learning, 47 h cage activity, open field exploration, a dark–light transition box, an accelerating rotarod, a stationary beam, a wire suspension task and a gait test) under normoxic and hypoxic conditions. No significant behavioural differences were found between WT and Ngb-overexpressing mice at three months old. However, one-year-old Ngb-overexpressing mice travelled more distance on the stationary beam compared with WT littermates. This result shows that the constitutive overexpression of Ngb might counteract the endogenous decrease of Ngb in crucial brain regions such as the cerebellum, thereby counteracting age-induced neuromotor dysfunction. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Contribution of endothelin to pulmonary vascular tone under normoxic and hypoxic conditions

The contribution of endothelin to resting pulmonary vascular tone and hypoxic pulmonary vasoconstriction in humans is unknown. We studied the hemodynamic effects of BQ-123, an endothelin type A receptor antagonist, on healthy volunteers exposed to normoxia and hypoxia. Hemodynamics were measured at room air and after 15 min of exposure to hypoxia (arterial PO(2) 99.8 +/- 1.8 and 49.4 +/- 0.4 mmHg, respectively). Measurements were then repeated in the presence of BQ-123. BQ-123 decreased pulmonary vascular resistance (PVR) 26% and systemic vascular resistance (SVR) 21%, whereas it increased cardiac output (CO) 22% (all P < 0.05). Hypoxia raised CO 28% and PVR 95%, whereas it reduced SVR 23% (all P < 0.01). During BQ-123 infusion, hypoxia increased CO 29% and PVR 97% and decreased SVR 22% (all P < 0.01). The pulmonary vasoconstrictive response to hypoxia was similar in the absence and presence of BQ-123 [P = not significant (NS)]. In vehicle-treated control subjects, hypoxic pulmonary vasoconstriction did not change with repeated exposure to hypoxia (P = NS). Endothelin contributes to basal pulmonary and systemic vascular tone during normoxia, but does not mediate the additional pulmonary vasoconstriction induced by acute hypoxia.     

Metabolic effects induced by epinephrine in Rana balcanica erythrocytes under normoxic and hypoxic conditions

The metabolic effects of epinephrine on Rana balacanica erythrocyte suspension were studied under normoxia and hypoxia. After epinephrine treatment, a 1.2-fold increase of lactate formation and a 20 per cent decrease of ATP concentration was found under normoxic conditions. These effects were rapid and specific to beta, alpha(1) and alpha(2) antagonists. Glycolysis was stimulated to almost the same extent by both epinephrine and forskolin as normoxic conditions. The stimulation of glycolysis was probably due to stimulation of phosphofructokinase (PFK) as well as to activation of Na(+), K(+)-ATPase. The decrease of ATP was a contributing factor to PFK activation. Despite the high levels of c-AMP at hypoxia, glycolysis was not further induced by epinephrine.     

Atrial natriuretic peptide during and after maximal and submaximal exercise under normoxic and hypoxic conditions

The present study was designed to investigate the influence of exercise intensity and duration as well as of inspiratory oxygen content on plasma atrial natriuretic peptide concentration [( ANP]) and furthermore to compare ANP with the effect on aldosterone concentration [( Aldo]). Ten untrained male subjects performed a maximal exercise test (ME) on a cycle ergometer and a submaximal test of 60-min duration at 60% of maximal performance (SE) under normoxia (N) and normobaric hypoxia (H) (partial pressure of oxygen: 12.3 kPa). Five subjects were exposed to hypoxia at rest for 90 min. The [ANP] was mostly affected by exercise intensity (5 min after ME-N, +298.1%, SEM 39.1%) and less by exercise duration (at the end of SE-N: +229.5%, SEM 33.2%). Hypoxia had no effect at rest and reduced the exercise response (ME-H, +184.3%, SEM 27.2%; SE-H, +172.4%, SEM 15.7%). In contrast to ANP, the Aldo response was affected more by duration at submaximal level (+290.1%, SEM 34.0%) than by short maximal exercise (+235.7%, SEM 22.2%). Exposure to hypoxia rapidly decreased [Aldo] (-28.5%, SEM 3.7% after 30 min, P less than 0.01), but did not influence the exercise effects (ME-H, +206.2%, SEM 26.4%; SE-H, +321.6%, SEM 51.6%). The [ANP] increase was faster than that of [Aldo] during the maximal tests and there was no difference during submaximal exercise. Changes in plasma volume (PV), sodium concentration, and osmolality (Osm) were most pronounced during maximal exercise (for ME-N: PV -13.1%, SD 3.6%, sodium +6.2 mmol.l-1, SD 2.7, Osm +18.4 mosmol.kg H2O-1, SD 6.5). Regression analysis showed high correlations between changes in [ANP] and in Osm during and after maximal exercise and between changes in [ANP] and heart rate for submaximal exercise. It is concluded that besides other mechanisms increased Osm might be involved in the exercise-dependent increase of plasma [ANP].     

Effect of exogenous CDP-choline on choline metabolism in isolated adult rat ventricular myocytes under normoxic and hypoxic conditions

The purpose of this study was to examine the effect of exogenous CDP-choline on choline metabolism and phosphatidylcholine biosynthesis in adult rat ventricular myocytes. Choline uptake and metabolism were examined, using [methyl³ H] choline. CDP-choline in the medium produced a concentration dependent reduction in the amount of radio-label in phosphocholine and phospholipid but it did not alter choline uptake into the myocytes. CDP-choline also did not antagonize the effect of hypoxia on phosphatidylcholine synthesis; rather it accentuated the hypoxia-induced reductions in cellular phosphocholine and phosphatidylcholine biosynthesis. These results indicate that the exogenous administration of CDP-choline alters choline metabolism in the heart by reducing the formation of phosphocholine and phosphatidylcholine without altering choline uptake and suggest an effect of a CDP-choline metabolite on choline metabolism which is not effective in opposing the effect of hypoxia on phosphatidylcholine biosynthesis.     

Proteomics profile of mesenchymal stromal cells and extracellular vesicles in normoxic and hypoxic conditions

Background aimsAlthough bone marrow-derived mesenchymal stromal cells (MSCs) have demonstrated success in pre-clinical studies, they have shown only mild therapeutic effects in clinical trials. Hypoxia pre-conditioning may optimize the performance of bone marrow-derived MSCs because it better reflects the physiological conditions of their origin. It is not known whether changes in the protein profile caused by hypoxia in MSCs can be extended to the extracellular vesicles (EVs) released from them. The aim of this study was to evaluate the proteomics profile of MSCs and their EVs under normoxic and hypoxic conditions.MethodsBone marrow-derived MSCs were isolated from six healthy male Wistar rats. After achieving 80% confluence, MSCs were subjected to normoxia (MSC-Norm) (21% oxygen, 5% carbon dioxide, 74% nitrogen) or hypoxia (MSC-Hyp) (1% oxygen, 5% carbon dioxide, 94% nitrogen) for 48 h. Cell viability and oxygen consumption rate were assessed. EVs were extracted from MSCs for each condition (EV-Norm and EV-Hyp) by ultracentrifugation. Total proteins were isolated from MSCs and EVs and prepared for mass spectrometry. EVs were characterized by nanoparticle tracking analysis. Proteomics data were analyzed by PatternLab 4.0, Search Tool for the Retrieval of Interacting Genes/Proteins, Gene Ontology, MetaboAnalyst and Reactome software.ResultsCell viability was higher in MSC-Hyp than MSC-Norm (P = 0.007). Basal respiration (P = 0.001), proton leak (P = 0.004) and maximal respiration (P = 0.014) were lower in MSC-Hyp than MSC-Norm, and no changes in adenosine triphosphate-linked and residual respiration were observed. The authors detected 2177 proteins in MSC-Hyp and MSC-Norm, of which 147 were identified in only MSC-Hyp and 512 were identified in only MSC-Norm. Furthermore, 718 proteins were identified in EV-Hyp and EV-Norm, of which 293 were detected in only EV-Hyp and 30 were detected in only EV-Norm. Both MSC-Hyp and EV-Hyp showed enrichment of pathways and biological processes related to glycolysis, the immune system and extracellular matrix organization.ConclusionsMSCs subjected to hypoxia showed changes in their survival and metabolic activity. In addition, MSCs under hypoxia released more EVs, and their content was related to expression of regulatory proteins of the immune system and extracellular matrix organization. Because of the upregulation of proteins involved in glycolysis, gluconeogenesis and glucose uptake during hypoxia, production of reactive oxygen species and expression of immunosuppressive properties may be affected.     

Pharmacokinetic / pharmacodynamic relationships of liposomal amphotericin B and miltefosine in experimental visceral leishmaniasis

BackgroundThere is a continued need to develop effective and safe treatments for visceral leishmaniasis (VL). Preclinical studies on pharmacokinetics and pharmacodynamics of anti-infective agents, such as anti-bacterials and anti-fungals, have provided valuable information in the development and dosing of these agents. The aim of this study was to characterise the pharmacokinetic and pharmacodynamic properties of the anti-leishmanial drugs AmBisome and miltefosine in a preclinical disease model of VL.Methodology / Principal findingsBALB/c mice were infected with L. donovani (MHOM/ET/67/HU3) amastigotes. Groups of mice were treated with miltefosine (orally, multi-dose regimen) or AmBisome (intravenously, single dose regimen) or left untreated as control groups. At set time points groups of mice were killed and plasma, livers and spleens harvested. For pharmacodynamics the hepatic parasite burden was determined microscopically from tissue impression smears. For pharmacokinetics drug concentrations were measured in plasma and whole tissue homogenates by LC-MS. Unbound drug concentrations were determined by rapid equilibrium dialysis. Doses exerting maximum anti-leishmanial effects were 40 mg/kg for AmBisome and 150 mg/kg (cumulatively) for miltefosine. AmBisome displayed a wider therapeutic range than miltefosine. Dose fractionation at a total dose of 2.5 mg/kg pointed towards concentration-dependent anti-leishmanial activity of AmBisome, favouring the administration of large doses infrequently. Protein binding was >99% for miltefosine and amphotericin B in plasma and tissue homogenates.Conclusion / SignificanceUsing a PK/PD approach we propose optimal dosing strategies for AmBisome. Additionally, we describe pharmacokinetic and pharmacodynamic properties of miltefosine and compare our findings in a preclinical disease model to available knowledge from studies in humans. This approach also presents a strategy for improved use of animal models in the drug development process for VL.     

Effect of isoproterenol on lipid metabolism and prostaglandin production in cultures of newborn rat heart cells, under normoxic and hypoxic conditions

Catecholamines are known to exert deleterious effects on heart cells and to provoke biochemical alterations similar to those observed during myocardial infarction. In order to investigate the mechanisms of these effects, we have studied in cultures of muscle (M) and fibroblast-like (F) cells derived from newborn rat hearts, the action of isoproterenol on membrane lipid metabolism and on prostaglandin production. We showed in F cells that beta-agonist stimulation produced a striking loss of membrane phospholipids and a moderate hydrolysis of cell triglycerides. In addition, isoproterenol treatment induced a significant stimulation of the secretion of prostacyclin but not of prostaglandin E2 by F cells. None of these effects were potentiated by oxygen deprivation. In contrast, M cells, which are sensitive to ischemia, failed to respond to isoproterenol treatment. These results suggest that catecholamines and hypoxia may exert combined deleterious effects on heart tissue by acting separately on the different target cells in vivo.     

Rhein inhibits angiogenesis and the viability of hormone-dependent and -independent cancer cells under normoxic or hypoxic conditions in vitro

Hypoxia is a hallmark of solid tumors, including breast cancer, and the extent of tumor hypoxia is associated with treatment resistance and poor prognosis. Considering the limited treatment of hypoxic tumor cells and hence a poor prognosis of breast cancer, the investigation of natural products as potential chemopreventive anti-angiogenic agents is of paramount interest. Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid), the primary anthraquinone in the roots of Cassia alata L., is a naturally occurring quinone which exhibits a variety of biologic activities including anti-cancer activity. However, the effect of rhein on endothelial or cancer cells under hypoxic conditions has never been delineated. Therefore, the aim of this study was to investigate whether rhein inhibits angiogenesis and the viability of hormone-dependent (MCF-7) or -independent (MDA-MB-435s) breast cancer cells in vitro under normoxic or hypoxic conditions. Rhein inhibited vascular endothelial growth factor (VEGF₁₆₅)-stimulated human umbilical vein endothelial cell (HUVEC) tube formation, proliferation and migration under normoxic and hypoxic conditions. In addition, rhein inhibited in vitro angiogenesis by suppressing the activation of phosphatidylinositol 3-kinase (PI3K), phosphorylated-AKT (p-AKT) and phosphorylated extracellular signal-regulated kinase (p-ERK) but showed no inhibitory effects on total AKT or ERK. Rhein dose-dependently inhibited the viability of MCF-7 and MDA-MB-435s breast cancer cells under normoxic or hypoxic conditions, and inhibited cell cycle in both cell lines. Furthermore, Western blotting demonstrated that rhein inhibited heat shock protein 90alpha (Hsp90α) activity to induce degradation of Hsp90 client proteins including nuclear factor-kappa B (NF-κB), COX-2, and HER-2. Rhein also inhibited the expression of hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF₁₆₅), epidermal growth factor (EGF), and the phosphorylation of inhibitor of NF-κB (I-κB) under normoxic or hypoxic conditions. Taken together, these data indicate that rhein is a promising anti-angiogenic compound for breast cancer cell viability and growth. Therefore, further studies including in vivo and pre-clinical need to be performed.     

Metabolic utilization of human osteoblast cell line hFOB 1.19 under normoxic and hypoxic conditions: A phenotypic microarray analysis

Osteoblasts play an important role in bone regeneration and repair. The hypoxia condition in bone occurs when bone undergoes fracture, and this will trigger a series of biochemical and mechanical changes to enable bone repair. Hence, it is interesting to observe the metabolites and metabolism changes when osteoblasts are exposed to hypoxic condition. This study has looked into the response of human osteoblast hFOB 1.19 under normoxic and hypoxic conditions by observing the cell growth and utilization of metabolites via Phenotype MicroArrays™ under these two different oxygen concentrations. The cell growth of hFOB 1.19 under hypoxic condition showed better growth compared to hFOB 1.19 under normal condition. In this study, osteoblast used glycolysis as the main pathway to produce energy as hFOB 1.19 in both hypoxic and normoxic conditions showed cell growth in well containing dextrin, glycogen, maltotriose, D-maltose, D-glucose-6-phospate, D-glucose, D-mannose, D-Turanose, D-fructose-6-phosphate, D-galactose, uridine, adenosine, inosine and α-keto-glutaric acid. In hypoxia, the cells have utilized additional metabolites such as α-D-glucose-1-phosphate and D-fructose, indicating possible activation of glycogen synthesis and glycogenolysis to metabolize α-D-glucose-1-phosphate. Meanwhile, during normoxia, D-L-α-glycerol phosphate was used, and this implies that the osteoblast may use glycerol-3-phosphate shuttle and oxidative phosphorylation to metabolize glycerol-3-phosphate.     

Effects of the ionophore grisorixin on myocardial function and metabolism in isolated perfused working rat heart under normoxic and hypoxic conditions

The effects of grisorixin, a monocarboxylic ionophore, were studied on isolated working rat hearts perfused with a suspension of washed pig erythrocytes (10% hematocrit). Grisorixin (2.5 microM) induced a transient stimulation of heart work, maximal at 5 min, expressed by an increase in heart rate (+21%) and aortic flow (+17%) and by an increase in coronary flow, maximal at 10 min (+47%). Concomitantly, myocardial Vo2 was slightly enhanced and the myocardial creatine phosphate level dropped (2 min). The lactate production increased by 82% (5 min) then dropped to the control value (10 min) and increased again till the 45th min (+211%), indicating a cardiac metabolic drift towards anaerobic glycolysis due to partial inhibition of the oxidative metabolism. Owing to its properties as an ionophore, grisorixin also induced a strong and rapid increase of potassium concentration in the perfusate and a decrease of sodium. Grisorixin was tested on hearts submitted to 20 min of hypoxic conditions. The hypoxia was rather mild and induced only very slight modifications of the ultrastructure. In the control series, heart rate and aortic flow decreased regularly while coronary flow and lactate production increased. Upon reoxygenation, the heart performances were rapidly restored. Grisorixin was administered according to four different protocols. When injected at the onset of hypoxia or 5 min later, it was able to maintain the aortic flow during the first minutes and induce a higher coronary dilation. These beneficial effects were short-lasting and no deleterious effects were found on the ultrastructure of hearts subjected to grisorixin whether after hypoxia or after reoxygenation.     

Development of the chick chorioallantoic capillary plexus under normoxic and normobaric hypoxic and hyperoxic conditions: a morphometric study.

Fertile eggs from the domestic fowl were incubated under normobaric normoxic (21% O2), hypoxic (14% O2), and hyperoxic (40% O2) conditions in order to examine the influence of the prevailing oxygen level on the growth and maturation of the chorioallantoic membrane. Eggs were sampled at regular stages throughout incubation for morphometric analysis. Under normoxic conditions, maturation of the capillary plexus occurred in two distinct stages, both of which contributed to a reduction in the thickness of the air-blood barrier. Between days 6 and 10, the capillaries sprouted and fused to form a dense plexus. Subsequently, between days 10 and 14, this plexus invaginated into the chorionic epithelium. Differentiation of the chorioallantoic membrane appeared maximal by the end of this period. Hypoxia resulted in diminished growth of the embryo and chorioallantoic membrane, but in accelerated maturation of the capillary plexus. Hyperoxia had a less marked effect but appeared to retard the final invagination of the plexus, resulting in a thicker air-blood barrier.     

Docosahexaenoic acid suppresses migration of triple-negative breast cancer cell through targeting metastasis-related genes and microRNA under normoxic and hypoxic conditions

There is insufficient evidence with respect to the effect of the standard anticancer therapeutic agents as well as common dietary supplements on the expression of such genes and microRNAs (miRNAs). Therefore, this study was aimed to study the effect of applying linoleic acid (LA) and docosahexaenoic acid (DHA) fatty acids alone or combined with Taxol on the expression of the matrix metalloproteinase (MMP)-9, MMP-2, vimentin, and talin2 genes, tumor-suppressor miR-194 and, onco-miR-106b in triple-negative breast cancer cell line, known as MDA-MB-231. MDA-MB-231 as metastatic breast cancer cell line was cultured and treated using 0.3 μM Taxol, 100 μM DHA, and 50 μM LA for 24 hours, alone or combined with Taxol under the normoxic and hypoxic conditions. Cells were harvested, after RNA extraction and complementary DNA synthesis, analysis of the expression levels of the studied genes and miRNAs was done through the use of the quantitative real-time polymerase chain reaction (qRT-PCR). Wound healing assay and Western blot analysis were also performed for confirmation. The results of qRT-PCR showed that treating the MDA-MB-231 cells with DHA caused an increase in the miR-194 expression and a decrease in the miR-106b expression, leading to the downregulation of the MMP-2 and MMP-9, and vimentin, and upregulation of the talin2 under the normoxic and hypoxic conditions. The results of the wound healing scratch assay revealed that the administration of the DHA and the DHA-Taxol combination caused the repression of cell migration in comparison with the control groups under the normoxic and hypoxic conditions. The results of the Western blot analysis demonstrated that DHA and the DHA-Taxol combination caused an increase in the expression of the talin2 protein rather than the control cells under both normoxic and hypoxic conditions. This study showed that DHA has significant antimetastatic effects against the triple-negative breast cancer cells. DHA could serve as a promising supplementation for suppressing the breast cancer cell migration, especially under the hypoxic condition.     

Beta-adrenoceptor-mediated cyclic AMP signal in different types of cultured nerve cells in normoxic and hypoxic conditions

β-adrenergic neurotransmission is an important factor regulating brain activity such as neuronal and glial survival, plasticity, membrane transport or cellular metabolism. Intracellular β-adrenergic signaling, via a stimulatory G protein (G_s), activates two major down-stream effectors, i.e., adenylyl cyclase (AC) and cAMP-dependent protein kinase A (PKA). The aim of this work was to study the ability of endogenous (adrenaline and noradrenaline) and exogenous (isoprenaline) β-adrenergic receptor agonists to increase cAMP in different types of nerve cells. Moreover, we wanted to precisely identify the receptor isoform involved in the observed phenomenon using selective β₁-, β₂- β₃-adrenoceptor blockers. In an additional study, the negative influence of hypoxia on the AC/cAMP intracellular signaling system was tested. The study was conducted in parallel on rat primary glial (astrocytes) cultures, primary neuronal cultures, C6 glioma cells and human T98G glioma cells. The formation of [³H] cAMP by agonists and antagonists was measured in [³H] adenine prelabeled cells under normoxic and hypoxic conditions. The obtained results revealed that adrenaline, noradrenaline and isoprenaline strongly stimulated cAMP production in all tested cell types (with highest potency in C6 glioma cells). In glial and neuronal cells the adrenaline-evoked cAMP effect was mediated mainly by the β₁-adrenoceptor, whereas in tumor cells the effect was probably mediated by all three β-subtype specific drugs. The AC/cAMP intracellular signaling system is affected by hypoxic conditions. Considering both physiological and therapeutic importance of β-family receptors the present work characterized the β-adrenoceptor-mediated cAMP signal transduction pathway in different nerve cells in normoxic and hypoxic conditions. The proposed in vitro model of hypoxic conditions may serve as a good model system to study the biological effects of endogenous catecholamines as well as potential therapeutics targeting adrenergic receptors, which are impaired during ischemia in vivo.     

Fungal volatilization of trivalent and pentavalent arsenic under laboratory conditions

Production of volatile derivatives of arsenic was studied using pure cultures of different fungal strains under laboratory conditions. Arsenic was used in its trivalent and pentavalent forms to evaluate the effect of arsenic valency on its biovolatilization. The average amount of volatilized arsenic for all fungal strains ranged from 0.026 mg to 0.257 mg and 0.024 mg to 0.191 mg of trivalent and pentavalent arsenic, respectively. These results show that approximately 23% of arsenic was volatilized from all culture media originally enriched with approximately 4 and 17 mg L(-1) of arsenic in trivalent form. The average amount of biovolatilized arsenic from culture media originally enriched with 4 and 17 mg L(-1) of arsenic in pentavalent form was 24% and 16%, respectively. The order of ability of arsenic biovolatilization is Neosartorya fischeri > Aspergillus clavatus > Aspergillus niger. Toxicity and fungal resistance to trivalent and pentavalent arsenic were also evaluated based on radial growth and biomass weight.