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Expression, purification, crystallization and preliminary X-ray diffraction analysis of an essential lipoprotein implicated in cell-wall biosynthesis in Mycobacteria.

Mycobacterium tuberculosis is a renewed cause of devastation in the developing world. Critical to the success of this re-emerging pathogen is its unusual waxy cell wall, which is rich in rare components including lipoarabinomannan (LAM) and its precursors, the phosphatidylinositol mannosides (PIMs). Balanced synthesis of these related glycolipids is intrinsic to both cell-wall integrity and virulence in M. tuberculosis and presents a promising, albeit poorly defined, therapeutic target. Here, the expression, purification and crystallization of an essential 600-amino-acid lipoprotein, LpqW, implicated in this process are reported. Crystals of LpqW were grown using 20-24%(w/v) PEG 4000, 8-16%(v/v) 2-propanol, 100 mM sodium citrate pH 5.5 and 10 mM DTT. A complete data set was collected at 2.4 A using synchrotron radiation on a crystal belonging to space group C222, with unit-cell parameters a = 188.57, b = 312.04, c = 104.15 A. Structure determination is under way.

Bacterial Proteins↗

Application of known X-ray phases in the crystallographic study of a small protein.

Phase information, assumed known from three-beam diffraction experiments, has been used successfully as input to direct methods to re-determine the crystal structure of rubredoxin. With data at 1.54 A resolution, starting sets containing 26 single phases, or alternatively 45 triplet phases, in both cases known with a mean error of +/-22.5 degrees, were sufficient to initiate solution of the structure. Conventional figures of merit were employed in the early stages to reject the majority of the incorrect phase models. The presence of a FeS(4) cluster in the structure was used in the interpretation of the initial maps. Phase sets including 2500 E's with a mean single phase error approximately 70 degrees or a mean triplet phase error approximately 80 degrees, both relative to the model from the crystallographic refinement, could be refined and expanded by Fourier recycling using the SAYTAN formalism. Several parameters have been varied to study their influence on phase expansion and refinement.

Journal Article↗

Common errors made by pathologists in the diagnosis of bone tumors.

The pathologist must obtain X-rays before attempting to make a diagnosis in a bone tumor. Errors are made with both benign and malignant lesions associated with calluses. Infection may stimulate a malignant bone tumor radiographically and pathologically. Secondary aneurysmal bone cyst may dominate the radiographic and pathologic findings and obscure the primary diagnosis. Osteoblastomas are becoming increasingly difficult to diagnose. The question of whether such an entity can become malignant has not been resolved. Cartilagenous tumors are often difficult to diagnose and the pathologist must not only rely on histologic findings, but in particular pay attention to the radiographic pattern which is often diagnostic. Accuracy of diagnosis in Ewing's sarcoma is essential because the treatment now can result in a cure rate of almost 40%. Certain lesions such as lymphoma or osteosarcoma can mimic this tumor. To re-emphasize the problem, the pathologist must have all the clinical information before attempting a diagnosis of a bone tumor and without the pertinent X-ray, errors are almost inevitable.

Adolescent↗

Rhenium(IV)-copper(II) heterobimetallic complexes with a bridge malonato ligand. Synthesis, crystal structure, and magnetic properties.

The Re(IV) complex [ReCl4(mal)]2-, in the form of two slightly different salts, (AsPh4)1.5(HNEt3)0.5[ReCl4(mal)] (1a) and (AsPh4)(HNEt3)[ReCl4(mal)] (1b), and the Re(IV)-Cu(II) bimetallic complexes [ReCl4(mu-mal)Cu(phen)2].CH3CN (2), [ReCl4(mu-mal)Cu(bpy)2] (3), and [ReCl4(mu-mal)Cu(terpy)] (4) (mal=malonate dianion, AsPh4=tetraphenylarsonium cation, HNEt3=triethylammonium cation, phen=1,10-phenanthroline, bpy=2,2'-bipyridine and terpy=2,2':6',2' '-terpyridine) have been synthesized and the structures of 1a, 1b, 2, and 3 determined by single-crystal X-ray diffraction. The structures of 1a and 1b are made up of discrete [ReCl4(mal)]2- anions and AsPh4+ and HNEt3+ cations, held together by electrostatic forces and hydrogen bonds. The Re(IV) atom is surrounded by four chloride anions and a bidentate malonate group, in a distorted octahedral environment. The structure of 2 consist of neutral dinuclear units [ReCl4(mu-mal)Cu(phen)2], with the metal ions united through a bridge carboxilato. The environment of Re(IV) is nearly identical to that in the mononuclear complex, and Cu(II) is five coordinate, being surrounded by four nitrogen atoms of two bidentate phen ligands and one oxygen atom of the malonato ligand. In 3, there are also dinuclear units, [ReCl4(mu-mal)Cu(bpy)2], but the Cu(II) ions complete a distorted octahedral coordination by binding with the free malonato oxygen atom of a neighbor unit, resulting in an infinite chain. The magnetic properties of 1-4 were also investigated in the temperature range 2.0-300 K. The magnetic behavior of 1a and 1b is as expected for a Re(IV) complex with a large value of the zero-field splitting (2D ca. 110 cm(-1)). For the bimetallic complexes, the magnetic coupling between Re(IV) and Cu(II) is antiferromagnetic in 2 (J=-0.39 cm(-1)), ferromagnetic in 4 (J=+1.51 cm(-1)), and nearly negligible in 3 (J=-0.09 cm(-1)).

Journal Article↗

Planar gamma scintigraphy--points to consider when quantifying pulmonary dry powder aerosol deposition.

Methodological aspects of planar gamma scintigraphy used to quantify pulmonary aerosol deposition were investigated using an experimental dry powder formulation. Particles of micronized salbutamol sulphate were labelled with technetium-99m and admixed to an ordered mixture of unlabelled micronized salbutamol sulphate and larger carrier particles of lactose. The radioaerosol was administered to 24 healthy subjects, 12 in each of two consecutive, similarly designed studies. Pulmonary deposition was determined using two methods: repeated planar imaging, and pharmacokinetic assessments following charcoal co-administration to prevent gastrointestinal salbutamol absorption. After due consideration had been taken to ensure appropriate radiolabelling, image acquisition and processing procedures, a scintigraphic estimate of 26.2% (24.2-28.4%) was obtained, which did not significantly differ from the pharmacokinetic estimate of 26.4% (24.4-28.7%). In summary, pre-study validation of the radiolabelling technique, quality control of radioaerosols produced during the study, correction for re-distribution of radiolabel from the lungs, selection of regions of interest, assessment of lung contours, correction for tissue attenuation of gamma rays and establishment of the actual recovery of radioactivity in the scintigraphic measurements could potentially affect the accuracy of the scintigraphic estimate of pulmonary deposition and, thus, should be carefully considered in the design or evaluation of any such study.

Administration, Inhalation↗

Planar gamma scintigraphy--points to consider when quantifying pulmonary dry powder aerosol deposition.

Methodological aspects of planar gamma scintigraphy used to quantify pulmonary aerosol deposition were investigated using an experimental dry powder formulation. Particles of micronized salbutamol sulphate were labelled with technetium-99m and admixed to an ordered mixture of unlabelled micronized salbutamol sulphate and larger carrier particles of lactose. The radioaerosol was administered to 24 healthy subjects, 12 in each of two consecutive, similarly designed studies. Pulmonary deposition was determined using two methods: repeated planar imaging, and pharmacokinetic assessments following charcoal co-administration to prevent gastrointestinal salbutamol absorption. After due consideration had been taken to ensure appropriate radiolabelling, image acquisition and processing procedures, a scintigraphic estimate of 26.2% (with 95% confidence interval of 24.2-28.4%) was obtained, which did not significantly differ from the pharmacokinetic estimate of 26.4% (24.4-28.7%). In summary, pre-study validation of the radiolabelling technique, quality control of radioaerosols produced during the study, correction for re-distribution of radiolabel from the lungs, selection of regions of interest, assessment of lung contours, correction for tissue attenuation of gamma rays and establishment of the actual recovery of radioactivity in the scintigraphic measurements could potentially affect the accuracy of the scintigraphic estimate of pulmonary deposition and, thus, should be carefully considered in the design or evaluation of any such study.

Administration, Inhalation↗

Reactivity of the organometallic fac-[(CO)3ReI(H2O)3]+ aquaion. Kinetic and thermodynamic properties of H2O substitution.

The water exchange process on [(CO)(3)Re(H(2)O)(3)](+) (1) was kinetically investigated by (17)O NMR. The acidity dependence of the observed rate constant k(obs) was analyzed with a two pathways model in which k(ex) (k(ex)(298) = (6.3 +/- 0.1) x 10(-3) s(-1)) and k(OH) (k(OH)(298)= 27 +/- 1 s(-1)) denote the water exchange rate constants on 1 and on the monohydroxo species [(CO)(3)Re(I)(H(2)O)(2)(OH)], respectively. The kinetic contribution of the basic form was proved to be significant only at [H(+)] < 3 x 10(-3) M. Above this limiting [H(+)] concentration, kinetic investigations can be unambiguously conducted on the triaqua cation (1). The variable temperature study has led to the determination of the activation parameters Delta H(++)(ex) = 90 +/- 3 kJ mol(-1), Delta S(++)(ex) = +14 +/- 10 J K(-1) mol(-1), the latter being indicative of a dissociative activation mode for the water exchange process. To support this assumption, water substitution reaction on 1 has been followed by (17)O/(1)H/(13)C/(19)F NMR with ligands of various nucleophilicities (TFA, Br(-), CH(3)CN, Hbipy(+), Hphen(+), DMS, TU). With unidentate ligands, except Br(-), the mono-, bi-, and tricomplexes were formed by water substitution. With bidentate ligands, bipy and phen, the chelate complexes [(CO)(3)Re(H(2)O)(bipy)]CF(3)SO(3) (2) and [(CO)(3)Re(H(2)O)(phen)](NO(3))(0.5)(CF(3)SO(3))(0.5).H(2)O (3) were isolated and X-ray characterized. For each ligand, the calculated interchange rate constants k'(i) (2.9 x 10(-3) (TFA) < k'(I) < 41.5 x 10(-3) (TU) s(-1)) were found in the same order as the water exchange rate constant k(ex), the S-donor ligands being slightly more reactive. This result is indicative of I(d) mechanism for water exchange and complex formation, since larger variations of k'(i) are expected for an associatively activated mechanism.

Journal Article↗

Rhenium diazenide ternary complexes with dithiocarbamate ligands: towards new rhenium radiopharmaceuticals.

The reaction of the mono-diazenide core, [ReCl2(NNC6H(4)-4-OCH3)(NCCH3)(PPh3)2], with four equivalents of the sodium or potassium salts of dithiocarbamate (dtc) ligands gives neutral complexes of the formula [Re(NNC6H(4)-4-OCH3)(dtc)2(PPh3)]. It is possible to use a wide range of dithiocarbamate ligands (S2CNRR') with a variety of R groups (R=R'=methyl, phenyl or ethyl; R=methyl, R'=phenyl and R=R'=morpholino). Substitution reactions with dtc ligands on the mono-diazenide derived from 2-hydrazinopyridine, [ReCl2(NNC5H4N)(PPh3)2, give the analogous complexes, [Re(NNC6H5N)(dtc)2(PPh3)]. The new complexes have been characterised by a combination of NMR spectroscopy, mass spectrometry and X-ray crystallography. Cyclic voltammetry measurements in dimethyl formamide show that the rhenium diazenido-dtc complexes undergo a quasi-reversible oxidation tentatively assigned to a ReIII/ReIV oxidation. Since the parent complex, [ReCl2(NNC6H(4)-4-R)(NCCH3)(PPh3)2] can be prepared directly from perrhenate and readily derivatised with dtc ligands these complexes have potential relevance to the development of new therapeutic rhenium radiopharmaceuticals.

Crystallography, X-Ray↗

Effects of hypercapnia on enhancement of decreased perfusion flow in non-infarcted brain tissues.

The effects of hypercapnia on enhancement of reduced cerebral perfusion were re-evaluated in areas of ischemia produced by occlusion of the canine middle cerebral artery. Perfusion was measured by 85Kr (beta-ray) and 133Xe (gamma-ray) clearances, fluorescein angiography and diameter measurement of arteries. Between 45 and 55 mm Hg of PaCO2 rCBF measured with both isotopes increased significantly. When PaCO2 was elevated above 55 mm Hg, there was a remarkable dissociation in the rCBF measured by both isotopes. Cortical blood flow measured by 85Kr clearance decreased and, conversely, rCBF measured by 133Xe continued to increase. Arteries of less than 50 mu in diameter in areas of ischemia dilated significantly during hypercapnia. At PaCO2 above 65 mm Hg, progressive sub-pial hemorrhage and extravasation of dye were observed as side effects of hypercapnia. The use of mannitol combined with hypercapnia appeared to be harmful. A PaCO2 level between 45 and 55 mm Hg increases perfusion in areas of mildly reduced rCBF.

Animals↗

Fatal stroke after completion pneumonectomy for torsion of left upper lobe following left lower lobectomy.

BACKGROUND: The lobar torsion after lung surgery is a rare complication with an incidence of 0.09 to 0.4 %. It may occur after twisting of the bronchovascular pedicle of the remaining lobe after lobectomy, usually on the right side. The 180-degree rotation of the pedicle produces an acute obstruction of the lobar bronchus (atelectasis) and of the lobar vessels as well. Without prompt treatment it progresses to lobar ischemia, pulmonary infarction and finally fatal gangrene. CASE PRESENTATION: A 62 years old female patient was admitted for surgical treatment of lung cancer. She underwent elective left lower lobectomy for squamous cell carcinoma (pT2 N0). The operation was unremarkable, and the patient was extubated in the operating room. After eight hours the patient established decrease of pO(2) and chest x-ray showed atelectasis of the lower lobe. To establish diagnosis, bronchoscopy was performed, demonstrating obstructed left lobar bronchus. The patient was re-intubated, and admitted to the operating room where reopening of the thoracotomy was performed. Lobar torsion was diagnosed, with the diaphragmatic surface of the upper lobe facing in an anterosuperior orientation. A completion pneumonectomy was performed. At the end of the procedure the patient developed a right pupil dilatation, presumably due to a cerebral embolism. A subsequent brain angio-CT scan established the diagnosis. She died at the intensive care unit 26 days later. CONCLUSION: The thoracic surgeon should suspect this rare early postoperative complication after any thoracic operation in every patient with atelectasis of the neighboring lobe. High index of suspicion and prompt diagnosis may prevent catastrophic consequences, such as, infarction or gangrene of the pulmonary lobe. During thoracic operations, especially whenever the lung or lobe hilum is full mobilized, fixation of the remaining lobe may prevent this life threatening complication.

Fatal Outcome↗

[An experimental study on the repair of the necrotic tubular bone with pedicled vascular periosteum grafting in rabbits].

OBJECTIVE: To investigate the mechanism of the repair of the necrotic tubular bone with pedicled vascular periosteum grafting in rabbits. METHODS: Twenty mature New Zealand rabbits aged 8 months were randomly divided into test and control groups by placebo-controlled protocol. A segment of radius measuring 1.2 cm in length with pedicled vascular periosteum of the rabbits in the test group was obtained from the middle distal part of the bone, and boiled for 30 minutes. The cooled bone was then re-grafted and wrapped with vascularized periosteum and immobilized, while those in the control group, no periosteal grafting was done. X-ray examinations of the bones were carried out at 2, 4, 6, 8 approximately 10 post operation weeks (POWs). Meanwhile 4-5 rabbits were sacrificed for the harvesting of the radius on both sides for histological examination. RESULTS: It was revealed by X-ray and histological study that the periosteal hyperplasia could be observed 2-4 weeks after operation in the test group. The sign of union of the fracture and the formation and reconstitution of the trabeculae could be observed 6-8 weeks after operation. The reconstitution of reticulated bone, the formation of the lamellar bone and new haversian system could be observed during 8-10 POW. The structure of the new formed bones became more mature and the fracture healed at 12 POW. In contrast, there was no changes in the necrotic bones in the control group at 2 POW and no signs of fracture healing at 4-6 POW, and the defect of the bones could be observed at 8 POW. The necrotic bones were totally dissolved and absorbed, and the bone defects could be observed clearly filled with fibrotic tissues at 12 POW. CONCLUSION: The necrotic bones could be replaced by regeneration of new bone originated from the pedicled vascular periosteum, so that the necrotic tubular bones could be repaired. But the necrotic bones in the control group were eventually absorbed, resulting in bone defects.

Animals↗

Synthesis of enantiopure oxorhenium(V) and arylimidorhenium(V) "3 + 2" Schiff base complexes. X-ray diffraction, cyclic voltammetry, UV-vis, and circular dichroism characterizations.

Two new oxorhenium(V) and two new arylimidorhenium(V) complexes of the Schiff base ligands 2-hydroxybenzaldehyde-((1R,2S)-1-amino-2-indanol)imine (1) (H(2)L(1)) and 3-(1-adamantyl)-2-hydroxy-5-methylbenzaldehyde-((1R,2S)-1-amino- 2-indanol)imine (2) (H(2)L(2)) have been prepared from the reaction of the precursor Re(O)(PPh(3))(2)Cl(3), Re(NC(6)H(5))(PPh(3))(2)Cl(3), or Re(NC(6)H(4)OCH(3))(PPh(3))(2)Cl(3) and the free ligands H(2)L(1,2). The complexes Re(O)(HL(1))(L(1)) (3), Re(O)(HL(2))(L(2)) (4), Re(NC(6)H(5))(HL(1))(L(1)) (5), and Re(NC(6)H(4)OCH(3))(HL(1))(L(1)) (6) have been isolated and fully characterized by IR, (1)H NMR, circular dichroism, LRMS-FAB, and elemental analysis. All the complexes have a chiral center at rhenium. A single enantiomer is obtained in all cases. Suitable crystals of 3 and 5 were used in X-ray structural determinations. Crystal data: (3) C(32)H(27)N(2)O(5)Re.CH(2)Cl(2), orthorhombic, P2(1)2(1)2(1), a = 9.5599(16) A, b = 9.9579(16) A, c = 31.712(5) A, V = 3018.9(9) A(3), T = 100(2) K, Z = 4. (5) C(40)H(38)N(3)O(5)Re, monoclinic, P2(1), a = 9.286(3) A, b = 18.759(6) A, c = 9.957(3) A, beta = 102.817(6) degrees, V = 1691.3(10) A(3), T = 100(2) K, Z = 2. The major characteristic of these complexes is the presence of two coordination modes for the Schiff base ligands on rhenium, a tridentate ligand (noted L(1,2)) and another bidentate ligand (noted HL(1,2)). In the latter, the -OH group of the indanol is free and tilts away from the coordination sphere. X-ray structural analyses in conjunction with circular dichroism were used to assign the absolute configuration at rhenium (C). Cyclic voltammetry, UV-vis, and circular dichroism data are presented and discussed. The complexes were found to be highly stable and to resist reduction even when treated with organic phosphanes.

Journal Article↗

Structure and physical properties of ternary rare-earth cobalt bismuth intermetallics (RE)12Co5Bi (RE = Y, Gd, Tb, Dy, Ho, Er, Tm).

The rare-earth intermetallic compounds (RE)12Co5Bi (RE = Y, Gd, Tb, Dy, Ho, Er, Tm) were prepared by arc-melting and annealing at 600 degrees C. These compounds extend the previously known (RE)6M2+xX1-x (M = Co, Ni; X = Ga, In, Sn, Pb) series with the Ho6Co2Ga-type structure to X = Bi. The crystal structure of Ho12Co5Bi was refined by the Rietveld method from powder X-ray diffraction data obtained using synchrotron radiation (Pearson symbol oI36, orthorhombic, space group Immm, Z = 2, a = 9.37598(14) A, b = 9.37871(14) A, c = 9.85465(13) A). Unlike other Ho6Co2Ga-type compounds, the 2a site in Ho12Co5Bi is exclusively occupied by Co atoms. Four-probe electrical resistivity measurements on sintered polycrystalline samples of (RE)12Co5Bi indicated metallic behavior. Magnetic measurements revealed behavior ranging from frequency-dependent maxima in the ac susceptibility for Y12Co5Bi to possible ferrimagnetic ordering for Gd12Co5Bi to antiferromagnetic ordering with metamagnetic transitions for the remaining compounds. As confirmed by band structure calculations using Y12Co5Bi as a model compound, Y-Y and Y-Co interactions are the most important bonding components, but matrix effects are likely responsible for anomalously short Co-Co contacts in the structure.

Journal Article↗

Should all patients with blunt trauma undergo 'routine' pelvic X-ray?

Routine pelvic radiography is carried out in all blunt trauma patients referred to our Emergency Department according to advanced trauma life support protocols. In this retrospective study, we questioned whether it is cost-effective to obtain 'routine' pelvic X-ray films. The notes and X-rays of all consecutive patients with blunt trauma who were referred to the Emergency Department of Dokuz Eylül University Hospital, Izmir, Turkey, between August and December, 1993 were re-evaluated. Eighteen patients who were unconscious and/or intoxicated and/or not fully orientated were excluded from the study. The remaining 65 patients were divided into three groups according to whether they were complaining of pain and whether pain was present on pelvic examination; group A consisted of those complaining of pain who also had pelvic pain on examination (28 patients), group B consisted of those complaining of pain or who had pain on pelvic examination (four patients) and group C consisted of those with no history of pain and no pain on pelvic examination (33 patients). Among the 65 patients whose pelvic X-rays were evaluated, 14 (22%) cases were shown to have fracture and all of these patients belonged to group A. The remaining 51 (78%) patients revealed no fractures. The difference between the groups were statistically significant (p < 0.001). As a result, we concluded that 'routine' pelvic radiography should be performed in the setting of blunt trauma only if the patient is: (a) unconscious, uncooperative or intoxicated, or (b) conscious, fully orientated and cooperative and complaining of pain both in the history and on pelvic examination. These limitations would allow us to decrease the amount of irradiation received, render more cost-effective treatment, and reduce the workload of emergency and radiology departments.

Adolescent↗

Structural changes in the region directly adjacent to the DNA-binding helix highlight a possible mechanism to explain the observed changes in the sequence-specific binding of winged helix proteins.

The hepatocyte nuclear factor 3 (HNF-3)/fork head (fkh) family contains a large number of transcription factors and folds into a winged helix motif. Despite having almost invariable amino acid sequences in their principal DNA-binding helices, HNF-3/fkh proteins show a wide diversity of sequence-specific binding. Previous studies of chimeric HNF-3/fkh proteins demonstrated that the binding specificity is primarily influenced by a region directly adjacent to the binding helix. We report our findings of an NMR structural study performed on an HNF-3/fkh family member (Genesis, formerly HFH-2) and compare it to that of another family member (HNF-3gamma) complexed to DNA and determined by X-ray crystallography. It is found that in comparison to HNF-3gamma, Genesis contains an extra small helix directly prior to the N terminus of the primary DNA contact helix. Due to the insertion of this helix, a shorter and slightly re-positioned primary DNA contact helix is observed, which we believe leads to the DNA-binding specificity differences among family members.

Binding Sites↗

Re-creating ancient hominid virtual endocasts by CT-scanning.

Probably the first radiographic study of human fossils, that by D. Gorganovic-Kramberger on Neandertal remains from Krapina, Croatia, was published in 1906, only 11 years after Röntgen announced the discovery of X-rays. Many subsequent studies on fossil hominids used regular clinical diagnostic radiological apparatus, as depicted in Atlas of Radiographs of Early Man by M.F. Skinner and G.H. Sperber (1982). Some specimens such as crania filled with heavily calcified matrix proved intractable. Ordinary radiographs of such specimens usually failed to reveal endocranial structure, as fossilized bone and calcified endocast were approximately equally radio-opaque. Thus, neither endocranial volume nor structural details were detectable. The only invasive method that could have been employed involved mechanical removal of the solid matrix, but this entailed hazards to the cranial vault and the destruction of the natural endocranial cast. In 1983--1984, G.C. Conroy and M. Vannier utilized recent advances in high-resolution computed tomography to produce non-invasive, intracranial capacity measurements of matrix-filled fossil skulls. They tried the method on two fossil mammal skulls filled with hard sandstone matrix (1984, Science 26:456-458), and then successfully applied it to a South African, matrix-filled cranium of the ancient hominid (hominin) species, Australopithecus africanus from Makapansgat (Conroy et al. 1990, Science 247:838-841). Details of the morphology of the endocranial surface of the braincase were revealed, including the pattern of venous sinus drainage in the posterior cranial fossa. A group based in St. Louis, Vienna, Paris, Rome, and Johannesburg has taken such studies further. Beautiful "virtual endocasts" have been produced on a large male specimen of A. africanus from Sterkfontein, South Africa, and the endocranial capacity has been determined (1998). The methods make it possible to re-create "virtual endocasts" of ancient hominids.

Cephalometry↗