Microbivores - Primary Phagocytic Systems

Primary Phagocytic Systems

The principal activity which drives microbivore scaling and design is the process of digestion of organic substances, which alsohas some similarity tothe digestion of food. The microbivore digestive system has four fundamental components -- an array of reversible binding sites to initially bind and trap target microbes, an array of telescoping grapples to manipulate the microbe, once trapped - a morcellation chamber in which the microbe is minced into small, easily digested pieces, and a digestion chamber where the small pieces are chemically digested.

Reversible Microbial Binding Sites

The first function the microbivore must perform is to acquire a pathogen to be digested. A collision between a bacterium of the target species and the nanorobotic device brings their surfaces into intimate contact, allowing reversible binding sites on the microbivore hull to recognize and weakly bind to the bacterium.

Bacterial membranes are quite distinctive, including such obvious markers as the family of outer-membrane trimeric channel proteins called porins in gram-negative bacteria like E. coli and other mic

surface proteins such as Staphylococcal protein A or endotoxin (lipopolysaccharide or LPS), a variable-size carbohydrate chain that is the major antigen of the outer membrane of gram-negative bacteria. Mycobacteria contain mycolic acid in their cell walls. And only bacteria employ right-handed amino acids in their cellular coats, which helps them resist attack by digestive enzymes in the stomach and by other organisms. Peptidoglycans, the main structural component of bacterial walls, are cross-linked with peptide bridges that contain several unusual nonprotein amino acids and D-enantiomeric forms of Ala, Glu, and Asp. D-alanine is the most abundant D-amino acid found in most peptidoglycans and the only one that is universally incorporated. Macrophages have evolved a variety of plasma membrane receptors that recognize conserved motifs having essential biological roles for pathogens, hence the surface motifs are not subject to high mutation rates; these pathogen receptors on macrophages have been called "pattern recognition receptors" and their targets "pathogen-associated molecular patterns". Genomic differences between virulent and non-pathogenic bacterial strains likely produce phenotypic differences that could enable the biasing of nanorobots towards the detection of the more toxic variants, if necessary.

Additionally, all bacteria of a given species express numerous unique proteins in their outermost coat. Some examples include:single-celled Staphylococcus aureus organism displays binding sites for human vitronectin on its surface; plasmid-specified major outer membrane protein TraTp of Escherichia coli is normally present at the cell surface; Streptococcus pyogenes (strain 6414) has surface binding sites to human collagen; another receptor protein specific to type II collagen (among the dozens of collagen types) are found on the surface of each Staphylococcus aureus (strain Cowan 1) cell. Researchers found that the same bacterial receptor would also specifically respond to synthetic collagen like analogs containing the peptide sequences (Pro-Gly-Pro)n, (Pro-Pro-Gly)10, and (Pro-OH-Pro-Gly)10) If the microbivore must distinguish among ~500 different bacterial species or strains, then each bacterial cell type may be uniquely identified using as few as log2(500) ~ 9 binary antigenic markers.

Assuming that nine species-specific bacterial coat ligands are sufficient to uniquely identify an encountered bacterium as belonging to the target species or strain, and that ~104 copies of each of the nine ligands are present on a bacterial surface of area ~10 micron2, then the mean distance between each ligand of the same type is 31.6 nm. A square array of 200 adjacent ligand receptors on the nanorobot surface, with each ligand or receptor active site ~5 nm2 in area (e.g., antibody-antigen complexes typically show contact interfaces of 6-9 nm2, involving 14-21 residues on each side, would on average overlap one such ligand that is resident in a bacterial surface pressed against it. If there are 100 such arrays uniformly distributed over the entire nanorobot surface, then a randomly chosen mutual contact area of only 1% of the nanorobot surface suffices to ensure that there is at least one array overlapping a unique ligand on the bacterial surface during a collision. Of course, the probability of binding, even given mutual contact, is not unity, but perhaps only ~10%. However, this factor is almost completely offset because there are nine equivalent array sets -- one set for each of the nine unique bacterial ligands -- and recognition and binding of any one of the nine unique ligands will suffice to bind the bacterium securely to the nanorobot.

Since array members need not be adjacent, the actual physical configuration on the microbivore surface is a bit different. The binding sites are modeled after the narrowband chemical sensor described below. sensor array

Each 3×3 receptor block consists of nine 7 nm × 7 nm receptor sites, one for each of the nine species-specific bacterial coat ligands. There are 20,000 of these 3×3 receptor blocks distributed uniformly across the microbivore surface. Each 3×3 receptor block measures 21 nm × 21 nm ×10 nm. A single receptor, if bound to a ligand, may provide a binding force of 40-160 pN, probably larger than the largest plausible in sanguo dislodgement force of ~100 pN and thus gripping the bacterium reasonably securely.

As an operational procedure, once any one of the nine key ligands has been detected, all of the remaining unoccupied receptors for that ligand in other receptor blocks can be deactivated, and so on until all nine ligands have been individually confirmed -- a combination lock whose completion triggers bacteriocide. Interestingly, during phagocytosis by macrophages most injected particles are recognized by more than one receptor; these receptors are capable of cross-talk and synergy, and phagocytic receptors can both activate and inhibit each other's function.

Microbial binding is energetically favored; if binding energy is ~240 zJ per microbial ligand (1 zeptojoule (zJ) = 10-21 J), then the power requirement for debinding a set of 9 occupied receptors in ~100 microsec is only ~0.02 pW.

Telescoping Grapples

Once the target bacterium has been confirmed and temporarily secured to the microbivore surface at >9 points with a minimum binding force of >360-1440 pN, telescoping robotic grapples emerge from silos in the nanodevice surface to establish secure anchorage to the microbe's plasma membrane or outer coat. Each grapple is mechanically equivalent to the telescoping robotic manipulator arm described by Drexler below

. This manipulator when fully extended is a cylinder 30 nm in diameter and 250 nm in length with a 150-nm diameter work envelope (to the microbivore hull surface), capable of motion up to 1 cm/sec at the tip at a mechanical power cost of ~0.6 pW at moderate load (or ~0.006 pW at 1 mm/sec tip speed), and capable of applying ~1000 pN forces with an elastic deflection of only ~0.1 nm at the tip.

Each telescoping grapple is housed beneath a self-cleaning arising cover mechanism that hides a vertical silo measuring 50 nm in diameter and 300 nm in depth, sufficient to accommodate elevator mechanisms needed to raise the grapple to full extension or to lower it into its fully stowed position. At a 1 mm/sec elevator velocity, the transition requires 0.25 millisec at a Stokes drag power cost (operating in human blood plasma) of 0.0008 pW, or 0.008 pW for 10 grapples maximally extended simultaneously . The elevator mechanism consists of compressed nitrogen gas rotored into or out of the subgrapple chamber volume from a small high-pressure sealed reservoir, a pneumatic piston providing the requisite extension or retraction force. A grapple-distension force of ~100 pN applied for a distance of 250 nm could be provided by 25 atm gas pressure in a minimum subgrapple chamber volume of 104 nm3, involving the importation of ~6000 gas molecules. Removal of these ~6000 gas molecules from a maximum subgrapple chamber volume of 105 nm3 provides a ~1 atm pressure differential and a maximum grapple-retraction force of ~100 pN; cables or other mechanisms may assist in retraction if more force is needed. The aperture of the arising silo cover can be controlled to continuously match the width of the protruding grapple, greatly reducing the intrusion of foreign biomolecules into the silo.

Each grapple is terminated with a reversible footpad ~20 nm in diameter. In the case of gram-positive bacteria, a footpad may consist of 100 close-packed lipophilic binding sites targeted to plasma membrane surface lipid molecules, providing a secure 100 pN anchorage between the nanorobot and the bacterium assuming a single-lipid extraction force of ~1 pN. In the case of gram-negative bacteria, a footpad with binding sites for ~3 murein-linked covalently attached transmembrane protein molecules would provide a secure 120-480 pN anchorage, assuming 40-160 pN/molecule and ~9 such molecules per 1000 nm2 of microbial surface. In either case, undesired adhesions with bacterial slime must be avoided. The footpad tool is rotated into, or out of, an exposed position from behind a protective cowling, using countercoiled internal pull cables.

The tiniest bacterium to be digested may be ~200 nm in diameter , but the smallest virus can be only ~16 nm wide . Since the work envelopes of adjacent grapples picking particles bound to the hull surface extend 150 nm toward each other from either side, the maximum center-to-center intergrapple separation that permits the ciliary transport of 16 nm objects is ~300 nm. This requires 1 grapple per 0.09 micron2 of nanorobot surface, for a total of 277 grapple silos uniformly distributed over the entire 26.885 micron2 microbivore outer hull, excluding the two 1-micron2 port doors. (One or more grapple-containing bridges across the annular exhaust port aperture may be necessary if it is desired to transport targets <200 nm in diameter from the circular DC exhaust port island to the main grapple field of the microbivore, allowing subsequent transport to the ingestion port inlet; such bridges are not included in the present design.) During transport, a bacterium of more typical size such as a 0.4 micron × 2 micron P. aeruginosa bacillus may be supported by up to 9 grapples simultaneously. A somewhat larger E. coli bacterium would be supported by up to 12 grapples.

After telescoping grapples are securely anchored to the captive bacterium, the receptor blocks are debonded from the microbial surface, leaving the grapples free to maneuver the pathogen as required. Grapple force sensors inform the onboard computer of the captive microbe's footprint size and orientation. The grapples then execute a ciliary transport protocol in which adjacent manipulators move forward and backward countercyclically, alternately binding and releasing the bacterium, with new grapples along the path ahead emerging from their silos as necessary and unused grapples in the path behind being stowed. Manipulator arrays, ciliary arrays (MEMS), and Intelligent Motion Surfaces are related precursor (and currently available) technologies.

Rodlike organisms are first repositioned to align their major axis perpendicular to a great circle plane containing both the device center point and the ingestion port at the front of the device. This keeps the organism traveling over surfaces having the largest possible radius of curvature during transport, thus minimizing any forces necessary to bend the bacterium as it follows the curved microbivore surface. To bend the microbe to the semimajor axis of the microbivore (Rcurve = 1.7 microns) requires F ~ 470 pN, or F ~ 800 pN for the semiminor axis (Rcurve = 1 micron), both of which are substantial bending forces in comparison to the nominal single-grapple anchorage force of 100-500 pN/footpad. Thus it is desirable to bend the bacterium as little as possible during transport. Bending forces may be minimized by adjusting grapple lengths to hold the bacillus farther from the microbivore surface near the endpoints of the footprint, and closer to the microbivore surface near the center of the footprint.

Organisms of all shapes are conveyed toward the ingestion port via cyclical ciliary cycling motions. At a transport velocity of 1 mm/sec, a microbe captured at the greatest possible distance from the ingestion port (~3 microns) is moved to the vicinity of the ingestion port in ~3 millisec. The Stokes law energy cost of transporting an E. coli bacterium through blood plasma side-on at 1 mm/sec is 0.01 pW, so transport power is dominated by mechanical losses in the grapples, a total of ~0.06 pW if 10 grapples are operated simultaneously.

Because the ingestion port is slightly recessed into the body of the nanorobot ellipsoid at the equator, the approaching bacterium must be carried around an inlet rim having a considerably smaller radius of curvature than the main body of the microbivore. The inlet rim is essential in this design and provides needed mechanical control from inlet-wall grapples as the microbe is fed into the ingestion port. From simple geometry, if one grapple is fully extended to length L = Lgrap and the adjacent grapple is almost fully retracted to length L ~ 0, then the bacillus can be conveyed around an inlet rim curve of radius Rrim with zero bending if the distance between the adjacent grapples is no more than dmax ~ 2 Rrim sin-1 (Lgrap / 2 Rrim)½ ~ 0.39 microns, taking Lgrap = 250 nm and Rrim ~ 0.25 microns at the inlet rim. This requires at least 1 grapple per dmax2 ~ 0.15 micron2 of nanorobot surface near the ingestion port, comfortably lower in number density than the 0.09 micron2/grapple elsewhere on the hull. Nevertheless, to ensure full control of the transported object near the ingestion port an additional 23 grapple silos are non-uniformly distributed over the 10% of microbivore surface nearest the ingestion port, sufficient to raise the mean number density to 0.05 micron2/grapple in that region. Thus there are a total of 300 grapple silos embedded in the entire microbivore outer hull, excluding the area covered by the two 1-micron2 port doors.

Ingestion Port and Morcellation Chamber

The ingestion port door is an oval-shaped irising mechanism with an elliptical aperture measuring 0.8654 microns × 1.4712 microns, providing a 1 micron2 aperture when fully open. Assuming 0.5 micron2 of contact surfaces sliding ~1 micron at 1 cm/sec, power dissipation is ~3 pW during the 0.1 millisec door opening or closing time. To allow handing small particles like viruses securely into the ingestion port, the porthole mechanism can be programmed to iris open in an off-center manner if required. For example, if manipulating a small virion particle the hole's center should initiate within 150 nm of a sidemost edge of the port (i.e., within one grapple surface-reach distance, either left or right side); after the growing aperture reaches the edge of the nearest side, it can then continue to dilate toward the edge on the opposite side while retaining its expanding elliptical shape. On the other hand, if a bacterium >~0.632 microns in diameter is being manipulated, the port door may be programmed to iris open from the center. During internalization the port doors perform gentle test-closings, with associated force sensors providing feedback as to the completeness of the internalization process and enabling the microbivore to detect the pinch points of linked bacilli to allow separation at these points, if necessary. In the case of motile bacilli having long flagellar tails, the premature closing of the ingestion port door may sever the tail, casting the immunogenic tail fragment adrift in the blood; this outcome must be avoided.

Opening the ingestion port door allows entry into the morcellation chamber (MC), a cylindrical chamber 2 microns in length and the same interior elliptical cross-section as the port door, giving a total open volume of 2 micron3 which is large enough to hold one intact microorganism because most sepsis-related bacteria are <2 micron3 in volume. Recessed into the MC walls are 10 diamondoid cutting blades (possibly multisegmented), each ~2 micron long, ~0.25 micron wide, and 10 nm thick with a 1 nm cutting edge, giving ~0.050 micron3 of blades (~0.005 micron3/blade).

Following the analysis of nano-morcellation systems described elsewhere [1], to mince material having Young's modulus ~108 N/m2 using one blade at a time (reserving the other 9 blades as replacements or to provide alternative chopping geometries) requires the application of ~100 nN/chop, consuming up to ~100 pW during a process in which the blade reciprocates at 50 Hz and travels at ~60 micron/sec, making 20 cuts in a total mincing time of 400 millisec. (Bacterial walls include a 3-6 nm thick hydrated sacculus and include a cross-linked peptidoglycan (murein) mesh with strands spaced ~1.3 nm apart .) The resulting morcellate should consist largely of organic chunks ~3-10 nm in diameter . An intriguing alternative configuration is a diamondoid sieve or dragnet that could be pulled repeatedly through the MC, analogous to pushing the microbe forcibly through a strainer.

Once microbial mincing is complete, the morcellate must be removed to the digestion chamber using an ejection piston. A 20-nm thick piston pusher plate driven by a 2 micron long, 10 nm thick pusher cable (energized by the chopping blade motor coupled through a mechanical transmission gearbox) comprises ~0.02 micron3 of device volume. This piston moves forward at ~20 microns/sec, applying ~1 atm of pressure to push morcellate of viscosity ~100 kg/m-sec through a 1 micron2 gated annular aperture for a chamber length of 2 microns, emptying the MC in ~100 millisec W. Interestingly, the energy dissipation rate required to disrupt the plasma membrane of ~95% of all animal cells transported in forced turbulent capillary flows is on the order of 108-109 W/m3, corresponding to a mechanical power input of 100-1000 pW into a 1 micron3 chamber volume. The annular MC/DC interchamber door must be opened before activating the MC ejection piston; its size and power specifications are similar to those of the annular DC exhaust port door.

The MC ejection piston also is used initially to draw the microbe into the MC in a controlled manner. By slowly pulling a vacuum after the ingestion port door has opened, the piston can apply ~1 atm of negative pressure over the ~1 micron2 leading surface of the bacterium, or up to ~100 nN of force. The Poiseuille flow of a microorganism of viscosity ~1000 kg/m-sec through a 1 micron2 aperture with a 1 atm pressure differential into a chamber 2 microns in length dissipates 0.2 pW as the bacterium is drawn into the chamber at a speed of 2 microns/sec, thus requiring ~1 second for complete internalization of 2 micron3 of ingesta.

Digestion Chamber and Exhaust Port

The digestion chamber (DC), like the MC, has a total open volume of 2 micron3. The DC is a cylinder of oval cross-section surrounding the MC, measuring roughly 2.0 microns in width, 1.3 microns in height, and 2.0 microns in length, with a mean ~0.5 micron clearance between the DC and MC walls and a materials volume of 0.11 micron3 assuming diamondoid walls ~10 nm thick. Morcellate is pumped from the MC into the DC where a preprogrammed sequence of engineered enzymes are successively injected and extracted, reducing the morcellate primarily to monoresidue amino acids, mononucleotides, free fatty acids and monosaccharides, which are then harmlessly discharged into the environment.

If the morcellate consists of organic chunks ~3-10 nm in diameter, enzymes directed against specific bond types may attack these bonds only if they are exposed on the outermost surface of each chunk. Considering for simplicity only proteinaceous chunks, and given that the average amino acid has a molecular weight of 141.1 daltons and a molecular volume of Vres ~ 0.49 nm3, then a chunk of volume Vchunk may be regarded as having Nlayer successive surface layers where Vchunk ~ Vres (1 + 2Nlayer)3. Taking Vchunk1/3 = 10.2 nm for the largest pieces implies a chunk comprised of 2197 residues and having Nlayer ~ 6 layers that must be processed sequentially, like peeling an onion one skin at a time. Thus the entire enzyme suite must be shuttled in and out of the DC six times, with one "layer" of all chunks being processed during each of the six subcycles.

Artificial Enzyme Suite

Artificial digestive enzymes may be designed to attack just one class of chemical bond. For example, the natural serine protease enzyme chymotrypsin only cleaves peptide bonds at the carboxylic ends of residues having large hydrophobic side chains, such as the aromatic amino acids phenylalanine, tryptophan, and tyrosine. The proteolytic enzyme trypsin exhibits a different specificity, cleaving peptide bonds on the C-terminal side of the basic residues arginine and lysine. The endopeptidase elastase attacks bonds adjacent to small amino acid residues such as alanine, glycine, and serine and will cleave tri-, tetra-, and penta-peptides of alanine. Enzymes which will cleave the unusual right-handed (D-enantiomeric) amino acids found in bacterial coats, including D-aminopeptidase or D-stereospecific amino-acid amidase, D-peptidase and DD-peptidase , carboxypeptidase DD and D-amino acid acylase are well-known.

To prevent self-digestion during storage and use, each artificial peptidase is engineered so that the class of residue it is designed to attack is not exposed on its own external physical surface -- that is, each artificial enzyme minimally exhibits strong autolysis resistance, with an ideal objective of near-zero autolysis. Another significant design constraint is that natural bacterial enzymes already present in the morcellate (e.g., elastase produced by P. aeruginosa) must have negligible activity against any of the microbivore's artificial enzymes. Since the target microbe's enzyme inventory is known in advance, the microbivore enzyme suite can be tailored to deal with any unusually troublesome bacterial enzymes, and optimal pH in the DC can be actively managed.

Ensuring biological digestive universality while allowing the enzyme engineer sufficient diversity of available protein building blocks requires a minimum of two pre-activated artificial enzymes that attack specific peptide bonds in each of the seven major amino acid classes -- acidic (Asn, Asp, Gln, Glu), aliphatic (Ala, Gly, Ile, Leu, Val), aromatic/hydrophobic (His, Phe, Trp, Tyr), basic (Arg, His, Lys), hydroxylic (Ser, Thr, Tyr), imino (Pro), and sulfur (Cys, Met). The present design thus includes a requirement for 14 artificial endopeptidases, plus 2 broad-spectrum artificial tripeptidase and dipeptidase if needed to complete the digestion of potentially bioactive tripeptides and dipeptides to free amino acids.

Enzymes capable of degrading nucleic acid polymers are classified as deoxyribonucleases (specificity for DNA) or ribonucleases (specifically hydrolyzing RNA), or as exonucleases (hydrolyzing a nucleotide only when present at a strand terminus, moving in only one direction, either 3'®5' or 5'®3') or endonucleases (cleaving internal phosphodiester bonds to produce either 3'-hydroxyl and 5'-phosphoryl termini or 5'-hydroxyl and 3'-phosphoryl termini) . Some endonucleases can hydrolyze both strands of a double-stranded molecule, others attack only one strand of a double-stranded molecule, while still others cleave only single-stranded molecules. Restriction endonucleases recognize specific DNA sequences -- for example, Hpa I recognizes a specific double-strand 6-base sequence (GTTAAC/CAATTG) and selectively cleaves both strands of the double strand in the middle at the TA/AT bond, producing an unreactive molecular "blunt end" . There are ten distinct dinucleotide bond combinations (AA, AC, AG, AT, CC, CG, CT, GG, GT, and TT), which suggests that 10 artificial endonucleases may suffice, plus 2 general-purpose dinucleases to complete the digestion to mononucleotides, for a total of 12 artificial polynucleotidases.

Additional engineered enzymes (not included in the present design) may be needed to digest bacteriophages that may be resident inside certain bacteria. To avoid digestion by bacterial restriction enzymes, phages often employ unusual molecular substitutions involving 2,6-diaminopurine, 6-methyladenine, 8-azaguanine, 5-hydroxymethyl uracil, 5-methylcytosine, 5-hydroxymethylcytosine, and others. For example, B. subtilis phage DNA replaces thymine with hydroxymethyluracil and uracil; S-2L cyanophage replaces adenine by 2-aminoadenine (2,6-diaminopurine); SPO1, SP82G, and Phi-e substitute hydroxymethyl dUTP for dTTP in the phage DNA up to 20%; PBS1 and PBS2 phages substitute uracil for thymine; T-even (T2/T4/T6) phage DNA replaces dCMP by hydroxymethylcytosine which is then further glycosylated, rendering the phage DNA resistant to host restriction; and in phage Mu DNA, a unique glycinamide moiety modifies about 15% of the adenine residues. Given our complete future knowledge of phage genomes and the bacteria they are likely to inhabit, a comprehensive phage digestive strategy can be planned and installed in advance, during microbivore design and construction. This problem is not considered serious in the case of standard antibiotic therapy.

Microbial lipids may be digested by analogs of pancreatic lipase (e.g., steapsin) or lipoprotein lipase which hydrolyze polyacylglycerols (mostly glycosyl diacylglycerols in bacteria) containing fatty acid chains into free fatty acids and glycerol, by cholesterol esterase that hydrolyzes cholesteryl esters into free cholesterol (although cholesterol and other sterols are relatively rare in microorganisms]), by phospholipase that attacks phospholipids producing glycerol, fatty acids, phosphoric acid, and perhaps choline, or by sphingolipidasesor ceramidases that hydrolyze the sphingolipids found in some bacteria, resulting in mostly glycerol and saturated (in bacteria) free fatty acids in the final digesta. Acyloxyacyl hydrolase removes the secondary (acyloxyacyl-linked) fatty acyl chains from the lipid A region of bacterial lipopolysaccharides (LPS endotoxin), thereby detoxifying the molecules. The present microbivore design assumes a requirement for 5 artificial lipases.

Microbial carbohydrates may be digested by an amylase that hydrolyzes starch and glycogen, and by a selection of oligosaccharidases (e.g., maltase, sucrase-isomaltase) and disaccharidases or saccharases (e.g., lactase, invertase, sucrase, trehalase) to complete the digestion to monosaccharides . (Lactase also has a second active site for splitting glycosylceramides .) The present design assumes a requirement for 4 artificial carbohydrases in the microbivore enzyme suite.

Finally, simple anions or cations may be required for pH management of the morcellate, and 25% of all enzymes contain tightly bound metal ions or require them for activity , most commonly Mg++, Mn++, Ca++, or K+; certain low-bioavailability but essential cofactors such as iron and copper might also need to be actively managed. It might also be necessary in some cases to inject and extract small quantities of superoxide dismutase, catalase and chelating agents such as metallothionein, ferritin, or transferrin to control potentially damaging concentrations of superoxides and metals in the morcellate, or small quantities of other specialized enzymes analogous to heme oxygenase, biliverdin reductase and beta-glucuronidases to digest bacterial porphyrins, enzymes to cleave bacterial rhodopsins, and so forth, but a full analysis of these factors is beyond the scope of this paper. The present design assumes a requirement for 3 additional chemical species of this type, to be manipulated simultaneously with the artificial enzymes as previously described.

Full digestion of the morcellate, constituting one complete digestion cycle, is thus presumed to require six subcycles of activity, with each subcycle involving the serial injection and extraction of 40 different enzymes or enzyme-related molecules (i.e., 40 sub-subcycles per subcycle), one after the other, for a total of 240 enzyme sub-subcycles. Interestingly, intracellular lysosomes are known to contain ~40 digestive enzymes capable of degrading all major classes of biological macromolecules -- including at least 5 phosphatases, 4 proteases, 2 nucleases, 6 lipases, 12 glycosidases, and an arylsulfatase.